Antiviral agents and uses thereof

ABSTRACT

The present invention relates to compounds which are found to exhibit an antiviral effect. The compounds are modulators of the activity of the viral haemagglutinin and/or neuraminidase enzymes.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/AU2015/050526, filed Sep. 7, 2015, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of NetherlandsApplication No. 2013420, filed Sep. 5, 2014, the contents of both ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the field of medical treatment. Moreparticularly, this invention relates to novel antiviral agents and theiruse in treating a disease or condition caused by a viral infection.

BACKGROUND TO THE INVENTION

Any reference to background art herein is not to be construed as anadmission that such art constitutes common general knowledge inAustralia or elsewhere.

Viruses are responsible for a wide range of mammalian disease whichrepresents a great cost to society. The effects of viral infection canrange from common flu symptoms to serious respiratory problems and canresult in death, particularly amongst the young, elderly andimmunocompromised members of the community.

Viruses of the family Orthomyxoviridae, including influenza virus typesA, B and C, and the family Paramyxoviridae are the pathogenic organismsresponsible for a significant number of human infections annually.

Taking the family Paramyxoviridae as one example, human parainfluenzaviruses types 1 to 3 (hPIV-1, 2 and 3) are the leading cause of upperand lower respiratory tract disease in infants and young children andalso impact the elderly and immunocompromised. Significantly, it isestimated that in the United States alone up to five million lowerrespiratory tract infections occur each year in children under 5 yearsold, and hPIV has been isolated in approximately one third of thesecases. There are currently neither vaccines nor specific antiviraltherapy to prevent or treat hPIV infections respectively, despitecontinuing efforts. Some of the more recent approaches have focussed onan entry blockade and the triggering of premature virus fusion by asmall molecule.

An initial interaction of the parainfluenza virus with the host cell isthrough its surface glycoprotein, haemagglutinin-neuraminidase (HN) andinvolves recognition of N-acetylneuraminic acid-containingglycoconjugates. The parainfluenza virus HN is a multifunctional proteinthat encompasses the functions of receptor binding (for cell adhesion)and receptor destruction (facilitating virus release), not only withinthe one protein, but apparently in a single binding site. In addition,the HN is involved in activation of the viral surface fusion (F) proteinnecessary to initiate infection of the target host cell. Inhibition ofhaemagglutinin and/or neuraminidase enzymes may therefore provide atarget for antivirals.

SUMMARY OF INVENTION

According to a first aspect of the invention, there is provided acompound of formula (I), or a pharmaceutically acceptable salt thereof:

wherein, R₁ is selected from the group consisting of COOH, or a saltthereof, C(O)NR₉R₁₀, C(O)OR₁₁ wherein R₉, R₁₀ and R₁₁ are independentlyselected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R₃ is selected from the group consisting of N-linked triazolesubstituted at one or both ring carbon atoms, optionally substitutedN-linked tetrazole, optionally substituted N-linked indole, optionallysubstituted N-linked isoindole, and optionally substituted N-linkedbenzotriazole;

R₄ is NHC(O)R₁₇ wherein R₁₇ is selected from the group consisting ofC₁-C₆ alkyl, C₁-C₆ haloalkyl and C₃-C₆ cycloalkyl;

R₆, R₇ and R₈ are independently selected from the group consisting ofOH, NH₂, C₁-C₆ alkyl, NR₁₈R₁₈′, C₁-C₆ alkoxy, —OC(O)R₁₈, —NH(C═O)R₁₈,and S(O)_(n)R₁₈, wherein n=0-2 and each R₁₈ and R₁₈′ are independentlyhydrogen or optionally substituted C₁-C₆ alkyl.

In one embodiment of the first aspect, the compound of formula (I) is acompound of formula (II):

wherein, R₁, R₃, R₄, R₆, R₇ and R₈ are as described above.

According to a second aspect of the invention there is provided apharmaceutical composition comprising an effective amount of a compoundof the first aspect, or a pharmaceutically acceptable salt thereof, anda pharmaceutically acceptable carrier, diluent and/or excipient.

Suitably, the pharmaceutical composition is for the treatment orprophylaxis of a disease, disorder or condition caused by viralinfection.

A third aspect of the invention resides in a method of treating adisease, disorder or condition caused by viral infection in a patientincluding the step of administering an effective amount of a compound ofthe first aspect, or a pharmaceutically effective salt thereof, or thepharmaceutical composition of the second aspect to the patient.

A fourth aspect of the invention provides for a compound of the firstaspect, or a pharmaceutically effective salt thereof, or thepharmaceutical composition of the second aspect for use in the treatmentof a disease, disorder or condition caused by viral infection in apatient.

A fifth aspect of the invention provides for use of a compound of thefirst aspect, or a pharmaceutically effective salt thereof, in themanufacture of a medicament for the treatment of a disease, disorder orcondition caused by viral infection.

In one embodiment of the third, fourth or fifth aspects, the disease,disorder or condition is influenza.

The influenza may be influenza A, B or C or parainfluenza.

In one embodiment, the parainfluenza is an hPIV-1, -2 or -3 virus.

Preferably, the patient is a domestic or livestock animal or a human.

A sixth aspect of the invention provides for a method of modulating theactivity of a viral haemagglutinin and/or neuraminidase enzyme includingthe step of contacting the enzyme with a compound of the first aspect.

The various features and embodiments of the present invention, referredto in individual sections above apply, as appropriate, to othersections, mutatis mutandis. Consequently features specified in onesection may be combined with features specified in other sections asappropriate.

Further features and advantages of the present invention will becomeapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be readily understood and put intopractical effect, preferred embodiments will now be described by way ofexample with reference to the accompanying figures wherein:

FIG. 1 shows the structures of N-acetylneuraminic acid (1), thesialidase inhibitor Neu5Ac2en (2), 4-azido-4-deoxy-Neu5Ac2en (3)zanamivir (4), the C-5 isobutyramido analogue of Neu5Ac2en (5), thereference hPIV inhibitor BCX 2798 (6), and the novel inhibitors 7-10;

FIG. 2 is a comparison of HI IC₅₀ values for inhibitors 6 and 10, usingguinea pig red blood cells (gp RBC, solid bar) and human red blood cells(h RBC, dashed bar);

FIG. 3 is a graphical representation of NI (solid) and HI (dashed) IC₅₀values for the Neu5Ac2en derivatives 2, 3, 5-10. Inhibitors 2, 3, 7, 8with a C5 acetamido group (left panel, group 1) and inhibitors 5, 6, 9,10 with a C5 isobutyramido group (right panel, group 2). Values are themeans of determinations from 3 independent experiments and error barscorrespond to calculated standard deviations;

FIG. 4A-C is a graphical comparison of NI (solid bar) and HI (dashedbar) IC₅₀ values for selected inhibitors. (A) Comparison of NI and HIIC₅₀ values for compounds 3, 6-10 and their C-4 hydroxyl analogues (2and 5). (B) Comparison of NI and HI IC₅₀ values for compounds 7-10 andtheir C-4 azido analogues (3 and 6). (C) Comparison of NI and HI IC₅₀values for compounds 6, 9 and 10 and their C-5 acetamido analogues (3, 7and 8 respectively);

FIG. 5 is (a) Titration (focus forming assay) of progeny virus after a48 h virus growth inhibition assay. Representative results of a progenyvirus titration. Virus was harvested after 48 h amplification in thepresence of 2 M of compounds 8, 10 or 6. Collected virus-culturesupernatants were diluted at least 1:1000 to make sure the remainingcompound has no effect on foci formation. (b) Virus growth inhibition ofthe reference inhibitor 6 and inhibitor 10. Virus growth inhibition wasdetermined by titration of progeny from a low MOI infected confluentLLC-MK2 monolayer in the presence of 2 μM inhibitor. At this inhibitorconcentration, 10 showed 94% inhibition compared with 14% inhibition for6. These results are representative of 2 independent experimentsperformed in duplicate and error bars correspond to the calculatedstandard deviation;

FIG. 6 shows Virus growth inhibition of the reference inhibitor 6 (□)and inhibitor 10 (▴) in various cell lines. Virus growth IC₅₀ values ofcompounds 6 (□) and 10 (▴) were determined by an in situ ELISA techniqueagainst both human cell lines (A549 and NHBE) and a monkey kidney cellline (LLC-MK2). IC₅₀ values of 54.6±3.8 μM and 2.1±0.6 μM (LLC-MK2);130.6±13.0 M and 10.3±0.3 μM (A549); 79.3±1.0 μM and 13.9±0.7 μM (NHBE)were determined for 6 and 10 respectively. These values were determinedfrom at least 2 independent experiments performed in triplicate anderror bars correspond to the calculated standard deviation;

FIG. 7 ¹H and STD NMR spectra of 8 in complex with hPIV-3 HN. (a) ¹H NMRspectrum of 8 and (b) STD NMR spectrum of 8 in the presence of 20 μMhPIV-3 HN at a protein-ligand ratio of 1:100 (2 mM 8). (c) ¹H NMRspectrum of the H7, H8, H9 and H9′ region. Signals from residualglycerol are marked as ★. (d) STD NMR spectrum of the H7, H8, H9 and H9′region. (e) The proposed binding epitope of 8;

FIG. 8 is a ¹H and STD NMR spectra and epitope map of 10 in complex withhPIV-3 HN. (a) ¹H NMR spectrum of 10. (b) STD NMR spectrum of 10 in thepresence of 20 μM hPIV-3 HN at a protein-ligand ratio of 1:100 (2 mM of10). (c) ¹H NMR spectrum of the H7, H8, H9 and H9′ region. (d) STD NMRspectrum of the H7, H8, H9 and H9′ region. (e) proposed binding epitopemap of inhibitor 10;

FIG. 9 is an STD NMR spectra comparison of 10 in complex with intacthPIV-3 virus or recombinant HN. (a) ¹H NMR spectrum of 10 in thepresence of hPIV-3 HN, (b) STD NMR spectrum of 10 in the presence ofintact hPIV-3 virus and (c) STD NMR spectrum of 10 in the presence ofhPIV-3 HN;

FIG. 10 is a superimposition of the phenyl protons from 10 in complexwith intact virus or recombinant HN. (a) ¹H NMR spectrum of 10 and (b)Superimpositions of STD NMR spectra from 10 in the presence of hPIV-3virus (black) or recombinant hPIV-3 HN (red);

FIG. 11 is a graphical representation of the results of cellcytotoxicity tests against A549 cells; and

FIG. 12 indicates the results of Neu2 inhibition assays for selectfluorinated and non-fluorinated compounds.

DETAILED DESCRIPTION

The present invention is predicated, at least in part, on the findingthat certain neuraminic acid derivatives display useful efficacy in thetreatment of diseases caused by viral infection. Particularly, thecompounds of the invention are useful in the inhibition of parainfluenzahaemagglutinin and/or neuraminidase enzymes.

Definitions

In this patent specification, the terms ‘comprises’, ‘comprising’,‘includes’, ‘including’, or similar terms are intended to mean anon-exclusive inclusion, such that a method or composition thatcomprises a list of elements does not include those elements solely, butmay well include other elements not listed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as would be commonly understood by those ofordinary skill in the art to which this invention belongs.

As used herein, “effective amount” refers to the administration of anamount of the relevant active agent sufficient to prevent the occurrenceof symptoms of the condition being treated, or to bring about a halt inthe worsening of symptoms or to treat and alleviate or at least reducethe severity of the symptoms. The effective amount will vary in a mannerwhich would be understood by a person of skill in the art with patientage, sex, weight etc. An appropriate dosage or dosage regime can beascertained through routine trial.

The term “pharmaceutically acceptable salt”, as used herein, refers tosalts which are toxicologically safe for systemic or localisedadministration such as salts prepared from pharmaceutically acceptablenon-toxic bases or acids including inorganic or organic bases andinorganic or organic acids. The pharmaceutically acceptable salts may beselected from the group including alkali and alkali earth, ammonium,aluminium, iron, amine, glucosamine, chloride, sulphate, sulphonate,bisulphate, nitrate, citrate, tartrate, bitarate, phosphate, carbonate,bicarbonate, malate, maleate, napsylate, fumarate, succinate, acetate,benzoate, terephthalate, palmoate, piperazine, pectinate and S-methylmethionine salts and the like.

The terms “substituted” and “optionally substituted” in each incidenceof its use herein, and in the absence of an explicit listing for anyparticular moiety, refers to substitution of the relevant moiety, forexample an alkyl chain or ring structure, with one or more groupsselected from C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, CN, OH, oxo,NH₂, Cl, F, Br, I, aryl and heterocyclyl which latter two may themselvesbe optionally substituted. When the term is used before the recitationof a number of functional groups then it is intended to apply to all ofthe listed functionalities unless otherwise apparent. For example,“optionally substituted amino, heterocyclic, aryl” means all of theamino, heterocyclic and aryl groups may be optionally substituted.

The term “alkyl” refers to a straight-chain or branched alkylsubstituent containing from, for example, 1 to about 12 carbon atoms,preferably 1 to about 8 carbon atoms, more preferably 1 to about 6carbon atoms, even more preferably from 1 to about 4 carbon atoms, stillyet more preferably from 1 to 2 carbon atoms. Examples of suchsubstituents include methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, 2-methylbutyl,3-methylbutyl, hexyl, heptyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, octyl, nonyl, decyl,undecyl, dodecyl and the like. The number of carbons referred to relatesto the carbon backbone and carbon branching but does not include carbonatoms belonging to any substituents, for example the carbon atoms of analkoxy substituent branching off the main carbon chain.

The term “cycloalkyl” refers to optionally substituted saturatedmono-cyclic, bicyclic or tricyclic carbon groups. Where appropriate, thecycloalkyl group may have a specified number of carbon atoms, forexample, C₃-C₆ cycloalkyl is a carbocyclic group having 3, 4, 5 or 6carbon atoms. Non-limiting examples may include cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyland the like.

The term “aryl” refers to an unsubstituted or substituted aromaticcarbocyclic substituent, as commonly understood in the art. It isunderstood that the term aryl applies to cyclic substituents that areplanar and comprise 4n+2π electrons, according to Hickel's Rule. C-6aryl is preferred.

The terms “heterocyclic” and “heterocyclyl” as used herein specificallyin relation to certain ‘R’ groups refers to a non-aromatic ring having 5to 7 atoms in the ring and of those atoms 1 to 4 are heteroatoms, saidring being isolated or fused to a second ring wherein said heteroatomsare independently selected from O, N and S. Heterocyclic includespartially and fully saturated heterocyclic groups. Heterocyclic systemsmay be attached to another moiety via any number of carbon atoms orheteroatoms of the radical and may be both saturated and unsaturated.Non-limiting examples of heterocyclic may be selected from pyrazole,imidazole, indole, isoindole, triazole, benzotriazole, tetrazole,pyrimidine, pyridine, pyrazine, diazine, triazine, tetrazine,pyrrolidinyl, pyrrolinyl, pyranyl, piperidinyl, piperazinyl,morpholinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolinyl,dithiolyl, oxathiolyl, dioxanyl, dioxinyl, oxazinyl, azepinyl,diazepinyl, thiazepinyl, oxepinyl and thiapinyl, imidazolinyl,thiomorpholinyl, and the like.

The term “protected OH” or “protected hydroxy” refers to a hydroxylgroup which is protected with a common protecting group such as an acylgroup, ether group or ester group including C₁-C₃ acyl, C₁-C₄ alkylgroups to form the ether or aryl, such as benzyl, forming the ether orC₁-C₄ ester.

The term “N-linked” as used herein with reference to compounds of thefirst aspect including compounds of formula (I) and (II), for example“N-linked triazole” or “N-linked heterocycle”, refers to the moietyattached at the C-4 position of the neuraminic acid core (R₃ in formula(I) and (II)) and limits that attachment to involving a directattachment between ring carbon and nitrogen atom. Preferably, it refersto the R₃ moiety being linked to the neuraminic acid core via a nitrogenatom which itself forms part of the appropriate heterocycle, such as oneof the nitrogens of a triazole ring, tetrazole, indole etc. For the“dilfuoro” compounds of formula (III) and (IIIa) the term N-linkedrefers to the R₃ moiety either being linked to the core via anintermediate nitrogen atom or, in the case of a heterocyclic moiety, itmay be via a nitrogen atom which forms part of the heterocycle itself,such as one of the nitrogens of a triazole ring

Whenever a range of the number of atoms in a structure is indicated(e.g., a C₁-C₁₂, C₁-C₁₀, C₁-C₉, C₁-C₆, C₁-C₄, alkyl, etc.), it isspecifically contemplated that any sub-range or individual number ofcarbon atoms falling within the indicated range also can be used. Thus,for instance, the recitation of a range of 1-12 carbon atoms (e.g.,C₁-C₁₂), 1-9 carbon atoms (e.g., C₁-C₉), 1-6 carbon atoms (e.g., C₁-C₆),1-4 carbon atoms (e.g., C₁-C₄), 1-3 carbon atoms (e.g., C₁-C₃), or 2-8carbon atoms (e.g., C₂-C₈) as used with respect to any chemical group(e.g., alkyl, etc.) referenced herein encompasses and specificallydescribes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12 carbon atoms, asappropriate, as well as any sub-range thereof (e.g., 1-2 carbon atoms,1-3 carbon atoms, 1-4 carbon atoms, 1-5 carbon atoms, 1-6 carbon atoms,1-7 carbon atoms, 1-8 carbon atoms, 1-9 carbon atoms, 1-10 carbon atoms,1-11 carbon atoms, 1-12 carbon atoms, 2-3 carbon atoms, 2-4 carbonatoms, 2-5 carbon atoms, 2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbonatoms, 2-9 carbon atoms, 2-10 carbon atoms, 2-11 carbon atoms, 2-12carbon atoms, 3-4 carbon atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7carbon atoms, 3-8 carbon atoms, 3-9 carbon atoms, 3-10 carbon atoms,3-11 carbon atoms, 3-12 carbon atoms, 4-5 carbon atoms, 4-6 carbonatoms, 4-7 carbon atoms, 4-8 carbon atoms, 4-9 carbon atoms, 4-10 carbonatoms, 4-11 carbon atoms, and/or 4-12 carbon atoms, etc., asappropriate).

As used herein, the terms “subject” or “individual” or “patient” mayrefer to any subject, particularly a vertebrate subject, and even moreparticularly a mammalian subject, for whom therapy is desired. Suitablevertebrate animals include, but are not restricted to, primates, avians,livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratorytest animals (e.g., rabbits, mice, rats, guinea pigs, hamsters),companion animals (e.g., cats, dogs) and captive wild animals (e.g.,foxes, deer, dingoes). A preferred subject is a human in need oftreatment for a disease or condition caused by viral infection. However,it will be understood that the aforementioned terms do not imply thatsymptoms are necessarily present.

According to a first aspect of the invention, there is provided acompound of formula (I), or a pharmaceutically acceptable salt thereof:

wherein, R₁ is selected from the group consisting of COOH, or a saltthereof, C(O)NR₉R₁₀, C(O)OR₁₁ wherein R₉, R₁₀ and R₁₁ are independentlyselected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R₃ is selected from the group consisting of N-linked triazolesubstituted at one or both ring carbon atoms, optionally substitutedN-linked tetrazole, optionally substituted N-linked indole, optionallysubstituted N-linked isoindole, and optionally substituted N-linkedbenzotriazole;

R₄ is NHC(O)R₁₇ wherein R₁₇ is selected from the group consisting ofC₁-C₆ alkyl, C₁-C₆ haloalkyl and C₃-C₆ cycloalkyl; and

R₆, R₇ and R₈ are independently selected from the group consisting ofOH, NH₂, C₁-C₆ alkyl, NR₁₈R₁₈′, C₁-C₆ alkoxy, —OC(O)R₁₈, —NH(C═O)R₁₈,and S(O)_(n)R₁₈, wherein n=0-2 and each R₁₈ and R₁₈′ are independentlyhydrogen or optionally substituted C₁-C₆ alkyl.

In one embodiment of the first aspect, the compound of formula (I) is acompound of formula (II):

wherein, R₁, R₃, R₄, R₆, R₇ and R₈ are as previously described.

Preferably, the triazole is a 1,2,3-triazole ring connected directly tothe neuraminic acid ring carbon at the N-1 position.

In one embodiment wherein the tetrazole is substituted, it issubstituted at the ring carbon only.

In one embodiment of the compound of formula (I) or (II) R₁ is COOH, ora salt thereof, or C(O)OR₁₁ wherein R₁₁ is selected from methyl, ethyland propyl.

In certain specific embodiments R₁ is selected from the group consistingof COOH, COONa and C(O)OMe.

In one embodiment of the compound of formula (I) or (II) R₃ is selectedfrom the group consisting of:

wherein, R₂₀ and R₂₁ are independently selected from the groupconsisting of hydrogen, hydroxyl, cyano, halo, C₁-C₆ alkyl, C₁-C₆haloalkyl, C₁-C₆ alkylether, optionally substituted pyridyl andoptionally substituted phenyl, and wherein at least one of R₂₀ and R₂₁is not hydrogen;

R₂₂ is selected from the group consisting of hydrogen, C₁-C₆ alkyl andoptionally substituted phenyl; and

R₂₃ and R₂₄ are independently selected from the group consisting ofhydrogen, hydroxyl, cyano, halo, C₁-C₆ alkyl and C₁-C₆ haloalkyl.

In certain preferred embodiments, R₂₀ and R₂₁ are selected from C₁-C₆alkyl, C₁-C₆ hydroxyalkyl, C₁-C₆ alkoxy, optionally substituted pyridyland optionally substituted phenyl.

In one embodiment, wherein when R₂₀, R₂₁ or R₂₂ are optionallysubstituted pyridyl or optionally substituted phenyl, as appropriate,then the substitution may be with a moiety selected from the groupconsisting of C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkylhydroxy, C₁-C₆alkoxy, halo, —C(O)OMe and —CH₂OCH₃.

In certain embodiments, R₃ may be selected from the group consisting of:

The specific moieties listed above may be combined with any disclosureof an R₁, R₄, R₆, R₇ or R₈ group as described herein.

In any of the aforedescribed embodiments, R₄ may be selected from thegroup consisting of:

In certain embodiments, R₄ is selected from the group consisting of—NHAc, —NHC(O)CH₂(CH₃)₂, —NHC(O)CF₃ and —NHC(O)CH₂CH₃.

In any embodiment of the compounds of formula (I) or (II), R₆, R₇ and R₈may be independently selected from the group consisting of OH, C₁-C₃alkoxy and —OC(O)R₁₈ wherein R₁₈ is optionally substituted C₁-C₃ alkyl.

In any one or more of the preceding embodiments, R₆, R₇ and R₈ may beindependently selected from OH and OAc.

In embodiments of formula (I) and formula (II) the compound may beselected from the group consisting of:

and all C-2 analogues thereof wherein the C-2 carboxy group is in theprotonated form, sodium salt form or C₁-C₃ ester prodrug form andwherein each compound may be considered to have close analoguesdisclosed wherein the R₄ position is explicitly replaced with any—NHC(O)R group wherein R is C₁-C₄ alkyl or haloalkyl.

It will be appreciated by a person of skill in the art of syntheticchemistry that the COOH group is easily interchanged with a salt form oran ester protecting group, for example a methyl ester group, and so allsuch forms are considered to be disclosed herein with reference to thecompounds listed above.

In one specific embodiment of formula (I) or formula (II), wherein R₄ isNHAc and R₃ is a substituted triazole then the triazole is notsubstituted with a carboxyl group.

In a further specific embodiment of formula (I) or formula (II), whereinR₄ is NHAc and R₃ is a triazole substituted only at the 4-position ofthe triazole ring (the 1-position being the ring nitrogen attached tothe neuraminic acid core) then the triazole is not substituted withpropyl, substituted propyl, substituted tert-butyl or diethoxyalkyl.

In one embodiment, the compound of the first aspect is a haemagglutininand/or neuraminidase modulator. Preferably, a haemagluttinin and/orneuraminidase inhibitor.

In one embodiment, it may be preferred that the haemagluttinin and/orneuraminidase inhibitor is an influenza or parainfluenza haemagluttininand/or neuraminidase inhibitor.

In one alternative aspect, referred to herein as the “difluoro” aspect,the invention resides in a compound of formula (III) or (IIIa), or apharmaceutically acceptable salt thereof:

wherein, R₁ is selected from the group consisting of COOH, or a saltthereof, C(O)NR₉R₁₀, C(O)OR₁₁, P(O)(OH)₂ and P(O)(OR)₂;

R_(1a) and R₂ are fluorine;

R₃ is selected from the group consisting of optionally substitutedN-linked heterocyclic, —NHC(O)NHR₁₂, —NHC(O)R₁₃ and —NHS(O)₂R₁₄;

R₄ is selected from the group consisting of NR₁₅R₁₆ and NHC(O)R₁₇; and

R₆, R₇ and R₈ are independently selected from the group consisting ofOH, NH₂, C₁-C₆ alkyl, NR₁₈R₁₈′, C₁-C₆ alkoxy, —OC(O)R₁₈, —NH(C═O)R₁₈,and S(O)_(n)R₁₈, wherein n=0-2 and each R₁₈ and R₁₈′ are independentlyhydrogen or optionally substituted C₁-C₆ alkyl.

In one embodiment of formula (III) or formula (IIIa) R₁ is selected fromthe group consisting of COOH, or a salt thereof, and COOR₁₁ wherein R₁₁is optionally substituted C₁-C₆ alkyl.

In one embodiment of formula (III) or formula (IIIa) R₁₁ is selectedfrom methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl and pentyl.

In one embodiment of formula (III) or formula (IIIa) R₁₂, R₁₃ and R₁₄are independently selected from the group consisting of optionallysubstituted benzyl and phenyl.

In one embodiment of formula (III) or formula (IIIa) R₃ is selected fromthe group consisting of pyrazole, imidazole, indole, isoindole,triazole, benzotriazole, tetrazole, pyrimidine, pyridine, pyrazine,diazine, triazine and tetrazine, all of which may be optionallysubstituted, and which are linked to the core via a ring nitrogen.

In one embodiment of formula (III) or formula (IIIa) R₃ is selected fromthe group consisting of:

In one embodiment of formula (III) or formula (IIIa) R₄ is selected fromthe group consisting of NR₁₅R₁₆ and NHC(O)R₁₇ and wherein R₁₅, R₁₆ andR₁₇ are independently selected from the group consisting of optionallysubstituted C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkenyl and C₃-C₆cycloalkyl.

In one embodiment of formula (III) or formula (IIIa) R₄ is NHC(O)R₁₇ andwherein R₁₇ is selected from the group consisting of optionallysubstituted C₁-C₆ alkyl, C₁-C₆ haloalkyl and C₃-C₆ cycloalkyl.

In one embodiment of formula (III) or formula (IIIa) R₄ is selected fromthe group consisting of:

In one embodiment of formula (III) or formula (IIIa) R₄ is selected from—NHC(O)-methyl, —NHC(O)-ethyl, —NHC(O)-propyl, —NHC(O)-isopropyl,—NHC(O)-n-butyl, —NHC(O)-sec-butyl, —NHC(O)-isobutyl, —NHC(O)-tert-butyland —NHC(O)-pentyl.

In one embodiment of formula (III) or formula (IIIa) R₅ is C₁-C₆ alkylor C₁-C₆ alkenyl each of which may be optionally substituted.

In one embodiment of formula (III) or formula (IIIa) R₅ is C₁-C₆ alkylsubstituted with hydroxy or protected hydroxy.

In one embodiment of formula (III) or formula (IIIa) R₆, R₇ and R₈ areindependently selected from OH, C₁-C₁₀ alkoxy and —OC(O)R₁₈ wherein R₁₈is C₁-C₁₀ alkyl.

In one embodiment of formula (III) or formula (IIIa) R₆, R₇ and R₈ areindependently selected from OH and OAc.

In one embodiment of the difluoro aspect, the compound of formula (III)or formula (IIIa), or a pharmaceutically acceptable salt thereof, isselected from the group consisting of:

wherein each incidence of COOH may be read interchangeably with COONa,and vice versa.

In any of the above embodiments of formula (III) or formula (IIIa) itmay be that R₃ is not N-linked aryl, that is, an aryl ring linked to thecore via an intermediate nitrogen atom.

It is postulated that compounds such as those represented by formula(III) and (IIIa), wherein there is a C-2 and 3 fluoro substitutionpattern, may be particularly efficacious against influenza strains.While not wishing to be bound by any particular theory it is believedsuch compounds are active by virtue of being effective inhibitors of theviral neuraminidase.

A number of synthetic pathways can be employed to access the compoundsof the invention. Scheme 1, below, shows one pathway by which certainknown neuraminidase inhibitors were synthesised to use as referencecompounds. Relevant synthetic techniques, which may also be applied tosynthesis of compounds of the first aspect, are disclosed in Carbohydr.Res. 244, 181-185 (1993); Carbohydr. Res. 342, 1636-1650 (2007); Bioorg.Med. Chem. Lett. 16, 5009-5013 (2006); and PCT application WO2002076971.

Scheme 2, below, shows a synthetic route used to access compounds 7-10which are preferred compounds of the first aspect.

In brief, the synthesis of the triazoles 7-10 was achieved using theknown 4-azido-4-deoxy-Neu5Ac2en based intermediates 11 and 12. Each ofthe two intermediates was exposed to either methylpropargyl ether orethynylbenzene under typical click azide-alkyne coupling conditions(heating a mixture of the 4-azido-4-deoxy-Neu5Ac2en derivative, alkyne,CuSO₄ and sodium ascorbate in a (1:1) mixture of water and tert-butanolfor 6 h) to afford the triazole derivative (FIG. 1). Triazoles 13 and 14(starting from 11) and the triazole derivatives 15 and 16 (starting from12) were isolated in yields of 78%, 82%, 71% and 84%, respectively. Theresulting per-O-acetylated triazole derivatives 13-16 were thendeprotected by treatment with aqueous methanol (50%) adjusted to pH13-14 at RT for 24 h to yield the final products 7-10 as sodium salts in85%, 96%, 92% and 89% yields, respectively.

The synthetic targets were, in part, driven by information gleaned frommolecular modelling of the hPIV-3 HN crystal structure. Particularly,the 216 loop of the hPIV-3 HN indicates significant flexibility and soit was postulated that Neu5Ac2en derivatives with somewhat bulky C4substituents could be accommodated in and lock open the 216 cavitywithin the active site. Molecular Dynamics (MD) simulations wereemployed to design and assess Neu5Ac2en derivatives that incorporate C4functionalised triazoles, as a base from which to test the theory. Fromthe initial study of 216 loop flexibility and the resultant 216 cavitydimensions, it was felt that relatively bulky C4 substituents on theunsaturated neuraminic acid-based template (compounds 7-10 shown inFIG. 1) could be well tolerated within the open 216 cavity. Furthermore,modelling of these C4 triazole substituted inhibitors in complex withhPIV-3 HN indicated that both an acetamido (7, 8) and an isobutyramido(9, 10) moiety at C5 on the template could also be well accommodatedsimultaneously within the C5 binding domain. It was assessed, using MDsimulations, the capacity of the bulkier triazole compounds (8 and 10)to efficiently lock open the 216 loop in hPIV-3 HN. The relativeinteraction energies of 8 and 10 in complex with hPIV-3 HN weredetermined to predict if the bulkier C5 acylamino moiety, in combinationwith the bulky C4 substituent, would be expected to improve inhibitoraffinity.

The methodology of the modelling and biological evaluation are discussedin detail in the experimental section but, briefly, led to a number ofconclusions concerning the influence of C4/C5 substituents on inhibitorpotency. Within each of the two screened groups, that is Group 1 (C5acetamido) and Group 2 (C5 isobutyramido), the order of potency based onthe substituent at C4 was found to be as follows:hydroxyl<azido≤4-methoxymethyltriazole<4-phenyltriazole. The weakestinhibition in both groups was observed for the 4-hydroxy derivatives 2and 5. This outcome supports the notion that the C4 binding domain,which accommodates the C4 hydroxyl group on Neu5Ac2en (2), hassignificant hydrophobic character and consequently does not favour theinteraction with a polar, hydrophilic group including a hydroxyl group.The hydrophobic nature of the pocket, combined with the large 216 cavitysize created by the opening of the 216 loop, does favour inhibitors,including inhibitors 8 and 10, that have the C4 hydroxyl group replacedwith bulky hydrophobic substituents.

Comparison of both group's IC₅₀ values revealed that replacement of theC5 acetamido group with an isobutyramido group in all of the preparedinhibitors led to overall enhanced potency. Typically, close to an orderof magnitude improvement was observed, except for the most potentinhibitor 10. Furthermore, analysis of the IC₅₀ values supports thenotion that the potency enhancement in the best inhibitors, 8 and 10,results predominantly from the introduction of the C4 substituent, withthe C5 substituent contributing to a much lesser extent. This notion isalso substantiated by STD NMR data analysis that led to an epitope mapof inhibitor 10 in which the protons of the 4-phenyltriazole moietyshowed the strongest contribution to the binding event of 10 in complexwith hPIV-3 HN, while the relative interactions observed for theisobutyramido group were less (50%).

The potent inhibition of both HN functions (NI and HI) by inhibitor 10demonstrates that the compound exerts its antiviral effect againsthPIV-3 by action on the virus' key HN protein. These findings arefurther supported by STD NMR experiments of 10 in complex with eitherintact virus or recombinant HN protein, that clearly show identical STDNMR signal intensities for the inhibitor's C4 triazole aromatic moiety.Moreover, the calculated binding epitope for 10 in complex with hPIV-3HN is in excellent agreement with the MD simulations that clearlypredict the close contact of the Neu5Ac2en derivative's H3 and the C4triazolo moiety's phenyl protons to the protein surface.

Furthermore, the in situ ELISA results are in good agreement with the NIand HI assay data. The LLC-MK2 cell-based assays demonstrate that 10 iseven more potent at the cellular level compared to NI and HIprotein-based assays. In this cell-based assay 10 was found to be ˜26times more potent than 6, whereas protein inhibition assays showed only˜8 and 11 fold improvement in NI and HI assays, respectively. Thisstrongly suggests 10 is a potent dual acting inhibitor that derivesefficient synergism from the inhibition of both the protein'sneuraminidase and haemagglutinin activities. This is in contrast to theknown inhibitor 6, which derives less synergistic effect as a result ofit's significantly poorer inhibition of the haemagglutinin activity.Finally, the extent of virus growth inhibition in both human cell linesfor inhibitor 10 compared with 6 clearly demonstrates the superiority ofthe designer ligand 10.

It will be appreciated that the compounds of the first aspect haveefficacy at more than just the hPIV-3 HN. It has been found, asindicated in the experimental section, that variations in the structureof the compound can tailor the activity to different hPIV or influenzaneuraminidases generally. For example, the difluoro derivativesdisclosed herein may be preferentially active against certain influenzaneuraminidases.

Particularly, the results indicate that not only is compound 10 a muchbetter inhibitor than prior art compound BCX-2798 (reference compound 6)in an inhibition assay to compare their capacity to inhibit hPIV-3 virusinfection and propagation in LLC-MK2 cells by a reduction of 94% versus14%, respectively, but other compounds of the first aspect have beenshown to have even greater potency than 10 or 6. For example, theinventors have designed, synthesised and biologically evaluatedcompounds IE1398-33 and IE927-99. These compounds have IC50 values forhPIV-3 HN of 1.97 micromolar and 0.27 micromolar, respectively. Thisrepresents a 1 to 2 order of magnitude improvement over the prior artreference compound BCX-2798. These results demonstrate the surprisinglevel of efficacy of the present compounds and hence the value of thepresent structure-guided inhibitor design.

According to a second aspect of the invention there is provided apharmaceutical composition comprising an effective amount of a compoundof formula (I), (II), (III) or (IIIa), or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier, diluent and/orexcipient.

Suitably, the pharmaceutical composition is for the treatment orprophylaxis of a disease, disorder or condition caused by viralinfection.

The pharmaceutical composition may include more than one compound offormula (I), (II), (III) or (IIIa). When the composition includes morethan one compound then the compounds may be in any ratio. Thecomposition may further comprise known co-actives, delivery vehicles oradjuvants.

The compound of formula (I), (II), (III) or (IIIa) is present in thepharmaceutical composition in an amount sufficient to inhibit orameliorate the disease, disorder or condition which is the subject oftreatment. Suitable dosage forms and rates of the compounds and thepharmaceutical compositions containing such may be readily determined bythose skilled in the art.

Dosage forms may include tablets, dispersions, suspensions, injections,solutions, syrups, troches, capsules and the like. These dosage formsmay also include injecting or implanting devices designed specificallyfor, or modified to, ensure placement at the site of connective tissuedegradation. A hydrogel is a preferred delivery form.

A third aspect of the invention resides in a method of treating adisease, disorder or condition caused by viral infection in a patientincluding the step of administering an effective amount of a compound offormula (I), (II), (III) or (IIIa), or a pharmaceutically effective saltthereof, or the pharmaceutical composition of the second aspect to thepatient.

A fourth aspect of the invention provides for a compound of formula (I),(II), (III) or (IIIa), or a pharmaceutically effective salt thereof, orthe pharmaceutical composition of the second aspect for use in thetreatment of a disease, disorder or condition caused by viral infectionin a patient.

A fifth aspect of the invention provides for use of a compound offormula (I), (II), (III) or (IIIa), or a pharmaceutically effective saltthereof, in the manufacture of a medicament for the treatment of adisease, disorder or condition caused by viral infection.

In one embodiment of the third, fourth or fifth aspects, the disease,disorder or condition is an infection caused by influenza orparainfluenza virus.

The infection may be caused by the influenza A, B or C or parainfluenzavirus.

In one embodiment, the parainfluenza is an hPIV-1, 2, 3 or 4 virus.

Preferably, the patient is a domestic or livestock animal or a human.

A sixth aspect of the invention provides for a method of modulating theactivity of a viral haemagglutinin and/or neuraminidase enzyme includingthe step of contacting the enzyme with a compound of formula (I), (II),(III) or (IIIa).

Preferably, the modulating involves inhibiting the viral haemagglutininand/or neuraminidase enzyme.

The following experimental section describes in more detail thecharacterisation of certain of the compounds of the invention and theirantiviral activity. The intention is to illustrate certain specificembodiments of the compounds of the invention and their efficacy withoutlimiting the invention in any way.

EXPERIMENTAL

Computational Chemistry

Molecular Dynamics simulations were performed with GROMOS software usingthe force-field parameter set 54A4 (ref 39). Initial coordinates weretaken from the X-ray structure (PDB accession code 1V3E) of hPIV-3 HN incomplex with 4 (FIG. 1). Compound 8 was superimposed on zanamivir (4)ring atoms from the crystal structure. Parameters for 8 were generatedin an analogous manner to existing parameters in the GROMOS force-field.The number of atoms in the final composite system for 1V3E-4 and IV3E-8was 78253 and 78084, respectively. Ionization states of amino acidresidues were assigned at pH 7.0. The histidine side chains wereprotonated at the NE-atom. Water molecules associated with the X-raystructure were removed, and replaced by explicit solvation using thesimple-point-charge (SPC) water model and periodic boundary conditions,consistent with previously published methodology. In the simulations,water molecules were added around the protein within a truncatedoctahedron with a minimum distance of 1.4 nm between the protein atomsand the square walls of the periodic box. All bonds were constrainedwith a geometric tolerance of 10⁻⁴ using the SHAKE algorithm.

A steepest-descent energy minimization of the system was performed torelax the solute-solvent contacts, while positionally restraining thesolute atoms using a harmonic interaction with a force constant of2.5×10⁴ kJ mol⁻¹ nm⁻². Next, steepest-descent energy minimization of thesystem without any restraints was performed to eliminate any residualstrain. The energy minimizations were terminated when the energy changeper step became smaller than 0.1 kJ mol⁻¹. For non-bonded interactions,a triple-range method with cut-off radii of 0.8/1.4 nm was used.Short-range van der Waals and electrostatic interactions were evaluatedat each time step, based on a charge-group pair-list. Medium-range vander Waals and electrostatic interactions, between (charge group) pairsat a distance longer than 0.8 nm and shorter than 1.4 nm, were evaluatedevery fifth time step, at which point the pair list was updated. Outsidethe longer cut-off radius a reaction-field approximation was used with arelative dielectric permittivity of 78.5. The centre of mass motion ofthe whole system was removed every 1000 time steps. Solvent and solutewere independently, weakly coupled to a temperature bath of 295 K with arelaxation time of 0.1 ps.

The systems were also weakly coupled to a pressure bath of 1 atm with arelaxation time of 0.5 ps and an isothermal compressibility of0.7513×10⁻³ (kJ mol⁻¹ nm⁻³)⁻¹. MD simulations of 20 ps periods withharmonic position restraining of the solute atoms and force constants of2.5×10⁴ kJ mol⁻¹ nm⁻², 2.5×10³ kJ mol⁻¹ nm⁻², 2.5×10² kJ mol⁻¹ nm⁻²,2.5×10¹ kJ mol⁻¹ nm⁻² were performed to further equilibrate the systemsat 50 K, 120 K, 1800 K, 240 K and 300 K, respectively. The simulationswere each carried out for 30 ns. The trajectory coordinates and energieswere saved every 0.5 ps for analysis. Simulation trajectories for hPIV-3HN in complex with 4 were produced in an analogous manner to thatdescribed above and were used for analysis and comparison to resultsobtained for hPIV-3 HN in complex with 8.

Analyses were done with the analysis software GROMOS++. Atom-positionalroot-mean-square differences (RMSDs) between structures were calculatedfor the residues comprising the 216-loop (residues 210-221) byperforming a rotational and translational atom-positional least-squaresfit of one structure on the second (reference) structure using a givenset of atoms (N, C_(α), C). Atom-positional root-mean-squarefluctuations (RMSFs) were calculated as an average from a 30 ns periodof simulation by performing a rotational and translationalatom-positional least-squares fit of the C_(α)-atoms of the trajectorystructures on the reference. RMSFs were calculated for all residuesincluding the 216-loop (residues 210-221). To obtain reduced,representative structural ensembles for the simulations, RMSD-basedconformational clustering was performed.

Structures extracted every 10 ps from simulations were superimposed onbackbone-C_(α) atoms to remove overall rotation and translation.Clustering of all atoms of residues that line the binding site (residues190-198, 210-221, 251-259, 274-280, 320-326, 334-339, 369-377, 407-413,474-480, 529-533) was performed to compare relative structuralpopulations of hPIV-3 HN protein from the different simulationtrajectories. The similarity criterion applied was the RMSD of all atomsof these residues with a cut-off of 0.13 nm. Final structures resultingfrom the 30 ns of MD simulations were extracted. Interaction energiesbetween hPIV-3 HN and inhibitors 8 and 10 were calculated using GROMOSgenerated energies, free-energy A-derivatives and block averages asseparate trajectory files, referred to as the energy trajectory. Theprogram ene_ana was used to extract individual interaction energy valuessuch as non-bonded contributions, i.e. van der Waals and Coulombinteractions from these files. Thus, these contributions between theligand and the protein were extracted from the energy trajectoryresulting from the simulation and interaction energies calculated. Theerror estimate was calculated from block averages of growing sizesextrapolating to infinite block size. Hydrophobic interactions wereanalysed and a map of interactions between inhibitor 10 and hPIV-3 HNwas created using LIGPLOT. To measure the extent of cavity opening forselected structures, the pocket volume was analysed using POVME.Importantly, extended simulation times, up to 80 ns provided outcomesentirely consistent with the data presented.

Compound 8 as a Model of a Neu5Ac2En-Based hPIV-3 HN Inhibitor with aBulky C4 Substituent

The simulation of the available hPIV-3 HN crystal structure (PDBaccession code 1V3E) in complex with 8 allowed an analysis of thedynamic behaviour of the protein relative to the zanamivir (4) boundstructure. Atom-positional root-mean-square deviations (RMSDs) of thehPIV-3 HN backbone atoms (C_(a), N, C) for the 216-loop from thesimulations of the hPIV-3 HN-4 and -8 complexes showed that the 216-loopundergoes more significant deviations from the crystal structure in thecase of the hPIV-3 HN-8 complex. RMSD-values of larger than 0.5 nm areobserved for the simulation of the hPIV-3 HN-8 complex, whereas thestructure deviates less (0.4 nm) for the hPIV-3 HN-4 complex. Thisnotion is further supported by the root-mean-square fluctuations (RMSFs)observed for the C_(a)-atoms of the backbone for residues associatedwith the 216-loop (residues 205-225). Increased RMS fluctuations areobserved for the residues of the second half of the 216-loop (215-220),where values of ˜0.3 nm are reached, indicating a substantialconformational rearrangement within that domain compared with thestarting hPIV-3 HN reference X-ray structure (PDB accession code IV3E).Table 1 shows a selection of RMSF values of residues comprised in the216-loop.

TABLE 1 Root-Mean-Square Fluctuations (RMSF) of selected residuescomprised in the 216-loop for the 1V3E-4 and 1V3E-8 simulated systems,in nanometers, compared with the reference X-ray structure IV3EReference X-ray Residue structure (IV3E) 1V3E-4 1V3E-8 210 0.036 0.0590.056 212 0.046 0.104 0.141 214 0.048 0.093 0.109 216 0.057 0.111 0.162218 0.060 0.134 0.289 220 0.041 0.070 0.079

The data suggests that loop flexibility, present under physiologicalsimulation conditions, has been significantly underestimated in crystalstructures and provides an opportunity for anti-parainfluenza virus drugdiscovery. Comparison of the hPIV-3 HN-4 complex and the hPIV-3 HN-8complex simulations demonstrates that the C4 substituent on 8 inducessignificant movement in the hPIV-3 HN 216-loop. The induced loop openingcould be seen from the solvent-accessible surface plots of the finalstructures obtained from 10 ns simulations of hPIV-3 HN-4 complex and 8.

The most populated conformational clusters from the MD simulations ofhPIV-3 HN in complex with 4 and 8 were identified and the superpositionof the final conformations from the simulations of hPIV-3 HN in complexwith 4 and 8 were generated. The difference in 216-loop conformationcould clearly be seen. The 216-loop cavity adopts a more openconformation when in complex with the more sterically-encumberedinhibitor 8. Generally, a wider cavity is observed for the simulation ofhPIV-3 HN-8 complex. The most populated cluster from the simulation ofthe hPIV-3 HN-4 complex has a slightly smaller cavity volume (654 Å³)compared to the simulated hPIV-3-8 complex (717 Å³).

To evaluate if a bulkier C5 acylamino moiety would be accommodated inthe presence of the C4 functionalised triazole an identical analysis of10 in complex with hPIV-3 HN was undertaken. This analysis indicatedthat a C5 isobutyramido moiety is well accommodated within the C5binding domain in the presence of the C4 functionalised triazole.

Relative Interaction Energies of 8 and 10 in Complex with hPIV-3 HN

To quantify the extent of inhibitor engagement with hPIV-3 HN an MDsimulations approach was used to determine theoretical averagedinteraction energies for the known inhibitor 2, as well as the novel C5acetamido and C5 isobutyramido inhibitors, 8 and 10 respectively.Average interaction energy (E_(avl)) values of −609.38±10.92 kJ mol⁻¹,−733.96±15.49 kJ mol⁻¹ and −821.88±10.93 kJ mol⁻¹ for 2, 8 and 10respectively in complex with hPIV-3 HN (1V3E)²⁵ were determined. Thesecalculations support the notion that the replacement of the acetamidomoiety in 8 with an isobutyramido moiety in 10 significantly improvesthe absolute E_(avl) value of the inhibitor in complex with the protein.Consequently, 10 is predicted to be a more potent hPIV-3 HN inhibitorthan 8. Further analysis of the MD simulation and extraction of thelowest (−1,078.13 kJ mol⁻¹) interaction energy structure of 10 incomplex with hPIV-3 HN (1V3E) revealed that 10 makes several keyinteractions within the binding pocket (FIG. 3d ). Noteworthy is theelectrostatic interaction between the ligand's carboxylate and thetriarginyl cluster (Arg192, Arg424, Arg502), hydrogen bond interactionsbetween the C7 hydroxyl group and Glu276 and the C5 isobutyramido NH andTyr337 and Glu409. Furthermore, additional hydrophobic interactions areobserved for both the C4 aromatic and C5 isobutyl functionalities,particularly with the peptide backbone, within the C4 and C5 bindingdomains respectively.

Chemistry

General Methods

Reagents and dry solvents were purchased from commercial sources andused without further purification. Anhydrous reactions were carried outunder an atmosphere of argon in oven-dried glassware. Reactions weremonitored using thin layer chromatography (TLC) on aluminium platespre-coated with Silica Gel 60 F254 (E. Merck). Developed plates wereobserved under UV light at 254 nm and then visualized after applicationof a solution of H₂SO₄ in EtOH (5% v/v) followed by charring. Flashchromatography was performed on Silica Gel 60 (0.040-0.063 mm) usingdistilled solvents. ¹H and ¹³C NMR spectra were recorded at 300 and 75.5MHz respectively on a BrukerAvance 300 MHz spectrometer. Chemical shifts(δ) are reported in parts per million, relative to the residual solventpeak as internal reference [CDCl₃: 7.26 (s) for ¹H, 77.0 (t) for ¹³C;DMSO: 2.50 (pent) for ¹H, 39.51 (hept) for ¹³C; D₂O: 4.79 (s) for ¹H].2D COSY and HSQC experiments were run to support assignments.Low-resolution mass spectra (LRMS) were recorded, in electrosprayionization mode, on a BrukerDaltonics Esquire 3000 ESI spectrometer,using positive mode. High-resolution mass spectrometry (HRMS) wererecorded for either the protected or deprotected final derivatives, andwere carried out by the University of Queensland FTMS Facility on aBrukerDaltonics Apex III 4.7e Fourier Transform micrOTOF-Q70 MS or bythe Griffith University FTMS Facility on a BrukerDaltonics Apex III 4.7eFourier Transform MS, fitted with an Apollo ESI source.

Final deprotected sialic acid derivatives were purified on a GracePure™SPE C18-Aq (5000 mg/20 mL) column using 2% acetonitrile/H₂O as asolvent. The purity of all synthetic intermediates after chromatographicpurification was determined to be >90% by ¹H and ¹³C NMR spectroscopyand the purity of reference compounds synthesised for screening purposes(2, 3, 5, 6), as well as the novel final products 7-10, was determinedto be 295%.

Synthesis

The synthesis of intermediates 11, 12 and 17-24 and reference inhibitors2, 3, 5, and 6 was achieved by the literature procedures. Generalmethods are set out in Schemes 1 and 2 which allow access to all of thecompounds described and synthesised herein.

General Procedure for the Synthesis of 18& 19:

A mixture of 17 or 11 (0.42 mmol), Boc₂O (275 mg, 1.27 mmol) and DMAP(50 mg, 0.42 mmol) in anhydrous THF (5 mL) was stirred under argonatmosphere at 60° C. o/n. After cooling to rt, the solvent wasevaporated under vacuum, and the residue was taken up in dichloromethane(DCM) for chromatographic separation on a silica gel column using ethylacetate:hexane (1:2) as solvent to yield pure 18 (170 mg, 71%) or 19(225 mg, 96%).

General Procedure for the Synthesis of 20 & 2:

To a methanolic solution of NaOMe, freshly prepared by dissolving sodiummetal (0.39 mmol, 9 mg) in anhydrous MeOH (5 mL), was added compound 18or 19 (0.26 mmol). The mixture was stirred at rt for 1 h and thenquenched with Amberlite® IR-120 (H+) resin (to pH=5). The resin wasfiltered off, washed with MeOH (5 mL×3) and the combined filtrate andwashings were evaporated under vacuum. The residue was re-dissolved inpyridine (2 mL), and acetic anhydride (0.5 mL) added. The reactionmixture was stirred at rt under argon atmosphere o/n and the solvent andexcess Ac₂O were then removed under vacuum. Finally, the residue wastaken up in DCM for chromatographic separation on a silica gel columnusing ethyl acetate:hexane (1:2) as solvent to yield pure 20 (112 mg,81%) or 21 (84 mg, 63%).

General Procedure for the Synthesis of 22 & 23:

To a solution of 20 or 21 (0.15 mmol) in anhydrous DCM (2 mL) was addedTFA (230 μL, 3.0 mmol) and the mixture was stirred at rt under argono/n. The reaction was diluted with DCM (20 mL) and quenched with sat.aq. NaHCO₃ solution (20 mL). The DCM layer was washed with water, brinethen dried over anhydrous Na₂SO₄. The dried organic solvent wasconcentrated under vacuum, and purified by silica gel chromatographyusing the suitable solvent system to yield pure 22 (58 mg, 90%) or 23(53 mg, 85%).

General Procedure for the Synthesis of 24 & 12:

To a solution of 22 or 23 (0.116 mmol) in DCM (2 mL) under argon wasadded Et₃N (82 μL, 0.58 mmol) and isobutyryl chloride (18 μL, 0.17mmol). The mixture was stirred at rt for 4 h and then loaded on a silicagel column for chromatographic separation using ethyl acetate:hexane(1:1) as solvent to yield pure 24 (50 mg, 84%) or 12 (51 mg, 91%).

General Procedure for the Synthesis of 5 & 6:

To a suspension of compound 24 or 12 (0.08 mmol) in a 1:1 mixture ofMeOH:H₂O (2 mL) at 0° C. was added dropwise a NaOH solution (1.0 M)until pH 14. The temperature was raised gradually to rt and the mixturewas stirred at rt overnight. The solution was then acidified withAmberlite® IR-120 (H+) resin (to pH=5), filtered and washed with MeOH(10 mL) and H₂O (10 mL). The combined filtrate and washings were thenconcentrated under vacuum and the residue was diluted with distilledwater (5 mL) and adjusted to pH=8.0 using 0.05 M NaOH to convert thecompound to its sodium salt. The compounds were then purified on aC18-GracePure™ cartridge using 2% acetonitrile/water as solvent to yieldpure 5 (26 mg, 94%) or 6 (24 mg, 82%) as fluffy white powders.

General Procedure for the Synthesis of 13-16:

The appropriate 4-azido-4-deoxy-Neu5Ac2en derivative (11 or 12, 0.22mmol) and the corresponding ethynyl derivative (0.33 mmol) weredissolved in a 1:1 mixture of tert-butanol:H₂O (4 mL). Copper(II)sulfate pentahydrate (4 mg, 0.015 mmol) was added to the mixturefollowed by sodium ascorbate (0.1 mL of freshly prepared 1 M solution inH₂O). The mixture was stirred at 45° C. for 6 h and then left to cool tort. The mixture was then diluted with DCM (100 mL), washed with 10%NH₄OH (50 mL), followed by brine (50 mL). The organic layer was driedover anhydrous Na₂SO₄ and concentrated under vacuum to give the crudeproducts 13-16, which were purified by silica gel chromatography usingan appropriate solvent system.

General Procedure for the Synthesis of 7-10:

To a suspension of the protected triazole derivative 13-16 in a 1:1mixture of MeOH:H₂O (2 mL) at 0° C. was added dropwise a NaOH solution(1.0 M) until pH ˜14. The temperature was gradually raised to rt and themixture was stirred at rt overnight. The solution was then acidifiedwith Amberlite® IR-120 (H⁺) resin (to pH=5), filtered and washed withMeOH (10 mL) and H₂O (10 mL). The combined filtrate and washings werethen concentrated under vacuum, then diluted with distilled water (5 mL)and adjusted to pH=8.0 using 0.05 M NaOH to convert the compound to itssodium salt. Finally, the compound was purified on a C18-GracePure™cartridge using 2% acetonitrile/water as solvent to yield the pureproducts 7-10.

Characterisation of Synthesised Compounds

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-methoxymethyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(13)

Purification by silica gel chromatography using ethyl acetate:acetone(6:1) yielded (90 mg, 78%) of pure 13. ¹H NMR (300 MHz, CDCl₃): δ 1.81(s, 3H, NAc), 2.05 (s, 6H, 2 OAc), 2.06 (s, 3H, OAc), 3.36 (s, 3H,OCH₃), 3.80 (s, 3H, COOCH₃), 4.17 (dd, J=12.5, 7.2 Hz, 1H, H-9), 4.29(m, 1H, H-5), 4.50 (s, 2H, OCH₂), 4.68-4.79 (m, 2H, H-9′, H-6), 5.40(ddd, J=7.4, 4.9, 2.5 Hz, 1H, H-8), 5.53 (dd, J=5.1, 1.8 Hz, 1H, H-7),5.78 (dd, J=10.0, 2.5 Hz, 1H, H-4), 6.00 (d, J=2.3 Hz, 1H, H-3), 7.05(d, J=9.1 Hz, 1H, NH), 7.64 (s, 1H, triazole-CH); ¹³C NMR (75 MHz,CDCl₃) δ 20.71, 20.79, 20.91 (3 OCOCH₃ ), 22.80 (NHCOCH₃ ), 48.39 (C-5),52.71 (COOCH₃ ), 58.16 (OCH₃), 58.38 (C-4), 62.21 (C-9), 65.68 (OCH₂),67.73 (C-7), 70.90 (C-8), 76.71 (C-6), 107.18 (C-3), 121.50(triazole-C-5), 145.24 (triazole-C-4), 145.92 (C-2), 161.27 (COOCH₃),170.06, 170.27, 170.81, 170.88 (NHCOCH₃, 3 OCOCH₃). LRMS [C₂₂H₃₀N₄O₁₁](m/z): (+ve ion mode) 549.1 [M+Na]⁺; HRMS (API) (m/z): [M+Na]⁺ calcd forC₂₂H₃₀N₄NaO₁₁ [M+Na]⁺549.1803. found, 549.1805.

Methyl7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(4-methoxymethyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(15)

Purification by silica gel chromatography using ethyl acetate:acetone(9:1) yielded (65 mg, 71%) of pure 15. ¹H NMR (300 MHz, CDCl₃): δ0.97(d, J=6.9 Hz, 3H, isobut-CH₃), 1.01 (d, J=6.8 Hz, 3H, isobut-CH₃), 2.05(s, 3H, OAc), 2.08 (s, 6H, 2 OAc), 2.24 (m, 1H, isobut-CH), 3.37 (s, 3H,OCH₃), 3.81 (s, 3H, COOCH₃), 4.14-4.29 (m, 2H, H-9, H-5), 4.51 (s, 2H,OCH₂), 4.68 (dd, J=12.5, 2.6 Hz, 1H, H-9′), 4.84 (dd, J=10.5, 1.7 Hz,1H, H-6), 5.38 (ddd, J=6.6, 5.5, 2.5 Hz, 1H, H-8), 5.48 (dd, J=5.5, 1.7Hz, 1H, H-7), 5.91 (dd, J=10.0, 2.4 Hz, 1H, H-4), 6.02 (d, J=2.4 Hz, 1H,H-3), 6.44 (d, J=8.7 Hz, 1H, NH), 7.59 (s, 1H, triazole-CH); ¹³C NMR (75MHz, CDCl₃): δ 18.81, 19.30 (isobut-2CH₃), 20.74, 20.90 (3 OCOCH₃ ),35.51 (isobut-CH), 48.79 (C-5), 52.69 (COOCH₃ ), 57.59 (C-4), 58.38(OCH₃), 62.08 (C-9), 65.73 (OCH₂), 67.66 (C-7), 70.79 (C-8), 76.24(C-6), 107.07 (C-3), 121.54 (triazole-C-5), 145.39 (triazole-C-4),145.82 (C-2), 161.30 (COOCH₃), 170.15, 170.23, 170.69 (3 OCOCH₃), 177.87(isobut-CO). LRMS [C₂₄H₃₄N₄O₁₁] (m/z): (+ve ion mode) 577.2 [M+Na]⁺.

Methyl7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(4-phenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(16)

Purification by silica gel chromatography using ethyl acetate:hexane(4:1) yielded (82 mg, 84%) of pure 16. ¹H NMR (300 MHz, CDCl₃): δ 0.95(d, J=6.8 Hz, 3H, isobut-CH₃), 0.99 (d, J=6.9 Hz, 3H, isobut-CH₃), 2.06(s, 3H, OAc), 2.09 (s, 6H, 2 OAc), 2.20-2.27 (m, 1H, isobut-CH), 3.83(s, 3H, COOCH₃), 4.16-4.39 (m, 2H, H-9, H-5), 4.70 (dd, J=12.5, 2.6 Hz,1H, H-9′), 4.88 (dd, J=10.5, 1.7 Hz, 1H, H-6), 5.40 (m, 1H, H-8), 5.52(dd, J=5.4, 1.7 Hz, 1H, H-7), 5.99 (dd, J=10.0, 2.4 Hz, 1H, H-4), 6.08(d, J=2.4 Hz, 1H, H-3), 6.51 (d, J=8.7 Hz, 1H, NH), 7.26-7.43 (m, 3H,Ph-H-3′, Ph-H-4′, Ph-H-5′), 7.74 (d, J=7.2 Hz, 2H, Ph-H-2′, Ph-H-6′),7.81 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, CDCl₃): δ 18.83, 19.31(isobut-2CH₃), 20.76, 20.92 (3 OCOCH₃ ), 35.55 (isobut-CH), 48.74 (C-5),52.72 (COOCH₃ ), 57.67 (C-4), 62.12 (C-9), 67.72 (C-7), 70.84 (C-8),76.39 (C-6), 107.25 (C-3), 118.84 (triazole-C-5), 125.83 (Ph), 128.47(Ph), 128.89 (Ph), 129.97 (Ph q carbon), 145.81 (C-2), 148.19(triazole-C-4), 161.35 (COOCH₃), 170.18, 170.26, 170.71 (3 OCOCH₃),178.00 (isobut-CO). LRMS [C₂₈H₃₄N₄O₁₀] (m/z): (+ve ion mode) 608.9[M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-methoxymethyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(7)

Yield=85%. ¹H NMR (300 MHz, D₂O): δ 1.84 (s, 3H, NAc), 3.31 (s, 3H,OCH₃), 3.52-3.71 (m, 2H, H-9 & H-7), 3.85 (dd, J=11.9, 2.6 Hz, 1H,H-9′), 3.95 (ddd, J=9.3, 6.2, 2.5 Hz, 1H, H-8), 4.33 (m, 1H, H-5), 4.51(dd, J=10.9, 1.2 Hz, 1H, H-6), 4.56 (s, 2H, OCH₂), 5.48 (dd, J=9.6, 2.3Hz, 1H, H-4), 5.80 (d, J=2.2 Hz, 1H, H-3), 8.08 (s, 1H, triazole-CH);¹³C NMR (75 MHz, D₂O): δ 21.65 (NHCOCH₃ ), 48.68 (C-5), 57.15 (OCH₃),59.94 (C-4), 63.06 (C-9), 64.22 (OCH₂), 68.05 (C-7), 69.71 (C-8), 75.34(C-6), 101.80 (C-3), 123.54 (triazole-C-5), 144.08 (triazole-C-4),150.43 (C-2), 168.75 (COONa), 173.57 (NHCOCH₃); LRMS [C₁₅H₂₁N₄NaO₈](m/z): (+ve ion mode) 432.1 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-phenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(8)

Yield=96%. ¹H NMR (300 MHz, D₂O): δ 1.87 (s, 3H, NAc), 3.64 (dd, J=12.1,6.4 Hz, 1H, H-9), 3.69 (dd, J=9.6, 1.4 Hz, 1H, H-7), 3.89 (dd, J=11.9,2.7 Hz, 1H, H-9′), 4.00 (ddd, J=9.3, 6.3, 2.7 Hz, 1H, H-8), 4.39 (m, 1H,H-5), 4.56 (dd, J=10.8, 1.4 Hz, 1H, H-6), 5.49 (dd, J=9.7, 2.3 Hz, 1H,H-4), 5.83 (d, J=2.2 Hz, 1H, H-3), 7.40 (m, 1H, Ph-H4′), 7.46 (dd,J=8.4, 6.9 Hz, 2H, Ph-H-3′, Ph-H-5′), 7.71 (d, J=7.1 Hz, 2H, Ph-H-2′,Ph-H-6′), 8.28 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, D₂O): δ 21.63(NHCOCH₃ ), 48.70 (C-5), 59.96 (C-4), 63.05 (C-9), 68.03 (C-7), 69.69(C-8), 75.31 (C-6), 101.75 (C-3), 120.41 (Ph), 125.61 (Ph), 128.77(triazole-C-5), 129.10 (Ph), 129.28 (Ph q carbon), 147.74(triazole-C-4), 150.48 (C-2), 168.75 (COONa), 173.58 (NHCOCH₃). LRMS[C₁₉H₂₁N₄NaO₇] (m/z): (+ve ion mode) 463.1 [M+Na]⁺; HRMS (API) (m/z):[M+1]⁺ calcd for C₁₉H₂₂N₄NaO₇ [M+H]⁺ 441.138070. found, 441.140189.

Sodium2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(4-methoxymethyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(9)

Yield=92%. ¹H NMR (300 MHz, D₂O): δ 0.98 (d, J=7.0 Hz, 3H, isobut-CH₃),1.03 (d, J=6.9 Hz, 3H, isobut-CH₃), 2.46 (m, 1H, isobut-CH), 3.39 (s,3H, OCH₃), 3.65-3.76 (m, 2H, H-9, H-7), 3.94 (dd, J=11.9, 2.7 Hz, 1H,H-9′), 4.04 (ddd, J=9.3, 6.3, 2.6 Hz, 1H, H-8), 4.49 (m, 1H, H-5),4.60-4.65 (m, 3H, H-6, OCH₂), 5.61 (dd, J=9.7, 2.3 Hz, 1H, H-4), 5.87(d, J=2.2 Hz, 1H, H-3), 8.18 (s, 1H, triazole-CH); ¹³C NMR (75 MHz,D₂O): δ 18.43 (isobut-CH₃), 18.64 (isobut-CH₃), 35.10 (isobut-CH), 48.19(C-5), 57.24 (OCH₃), 59.86 (C-4), 63.07 (C-9), 64.24 (OCH₂), 68.13(C-7), 69.82 (C-8), 75.43 (C-6), 102.02 (C-3), 123.65 (triazole-C-5),144.07 (triazole-C-4), 150.30 (C-2), 168.81 (COONa), 180.66 (isobut-CO).LRMS [C₁₇H₂₅N₄NaO₈] (m/z): (+ve ion mode) 459.0 [M+Na]⁺; HRMS (API)(m/z): [M+Na]⁺ calcd for C₁₇H₂₅N₄Na₂O₈[M+Na]⁺459.1462. found, 459.1458.

Sodium2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(4-phenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(10)

Yield=89%. ¹H NMR (300 MHz, D₂O): δ 0.94 (d, J=6.9 Hz, 3H, isobut-CH₃),0.99 (d, J=6.9 Hz, 3H, isobut-CH₃), 2.43 (m, 1H, isobut-CH), 3.60-3.76(m, 2H, H-9, H-7), 3.93 (dd, J=12.0, 2.7 Hz, 1H, H-9′), 4.04 (ddd,J=9.2, 6.3, 2.6 Hz, 1H, H-8), 4.51 (m, 1H, H-5), 4.62 (d, J=11.0 Hz, 1H,H6), 5.58 (dd, J=9.7, 2.3 Hz, 1H, H-4), 5.88 (d, J=2.2 Hz, 1H, H-3),7.42-7.54 (m, 3H, Ph-H-3′, Ph-H-4′, Ph-H-5′), 7.78 (d, J=7.1 Hz, 2H,Ph-H-2′, Ph-H-6′), 8.36 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, D₂O): δ18.38 (isobut-CH₃), 18.65 (isobut-CH₃), 35.10 (isobut-CH), 48.23 (C-5),59.91 (C-4), 63.07 (C-9), 68.15 (C-7), 69.76 (C-8), 75.41 (C-6), 101.96(C-3), 120.66 (Ph), 125.67 (Ph), 128.81 (triazole-C-5), 129.16 (Ph),129.36 (Ph q carbon), 147.71 (triazole-C-4), 150.32 (C-2), 168.80(COONa), 180.67 (isobut-CO). LRMS [C₂₁H₂₅N₄NaO₇] (m/z): (+ve ion mode)491.2 [M+Na]⁺; HRMS (API) (m/z): [M+Na]⁺ calcd forC₂₁H₂₅N₄Na₂O₇[M+Na]⁺491.1513. found, 491.1515.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-isobutyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-6)

Purification by silica gel chromatography using ethylacetate yielded (93mg, 66%) of pure IE832-6. ¹H NMR (300 MHz, CDCl₃): δ 0.89 (d, J=6.6 Hz,6H, isobutyl-2CH₃), 1.81 (s, 3H, NAc), 1.93 (m, 1H, isobutyl-CH), 2.06(s, 6H, 2 OAc), 2.09 (s, 3H, OAc), 2.58 (d, J=6.9 Hz, 2H, isobutyl-CH₂),3.82 (s, 3H, COOCH₃), 4.17 (dd, J=12.5, 6.7 Hz, 1H, H-9), 4.38 (m, 1H,H-5), 4.67 (dd, J=12.4, 2.3 Hz, 1H, H-9′), 4.77 (d, J=10.5 Hz, 1H, H-6),5.40 (m, 1H, H-8), 5.55 (d, J=5.4 Hz, 1H, H-7), 5.88 (d, J=9.9 Hz, 1H,H-4), 6.03 (s, 1H, H-3), 7.07 (brs, 1H, NH), 7.49 (s, 1H, triazole-CH);¹³H NMR (75 MHz, CDCl₃): δ 20.74, 20.81, 20.93 (3 OCOCH₃ ), 22.12, 22.84(2 isobutyl-CH₃+NHCOCH₃ ), 28.60 (isobutyl-CH), 34.17 (isobutyl-CH₂),48.04 (C-5), 52.76 (COOCH ₃), 58.62 (C-4), 62.06 (C-9), 67.56 (C-7),70.55 (C-8), 76.57 (C-6), 106.82 (C-3), 120.94 (triazole-C-5), 146.03(C-2), 146.83 (triazole-C-4), 161.30 (COOCH₃), 169.99, 170.00 170.13,170.78 (NHCOCH₃, 3 OCOCH₃). LRMS [C₂₄H₃₄N₄O₁₀] (m/z): (+ve ion mode)561.2 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-isobutyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-8)

Yield=79%. ¹H NMR (300 MHz, D₂O): δ 0.82 (d, J=6.6 Hz, 6H,isobutyl-2CH₃), 1.81-1.86 (m, 4H, NAc & isobutyl-CH), 2.53 (d, J=6.9 Hz,2H, isobutyl-CH₂), 3.54-3.68 (m, 2H, H-9 & H-7), 3.85 (dd, J=11.9, 2.6Hz, 1H, H9′), 3.94 (ddd, J=9.3, 6.3, 2.5 Hz, 1H, H-8), 4.33 (m, 1H,H-5), 4.49 (dd, J=10.9, 1.2 Hz, 1H, H-6), 5.40 (dd, J=9.7, 2.2 Hz, 1H,H-4), 5.78 (d, J=2.2 Hz, 1H, H-3), 7.80 (s, 1H, triazole-CH); ¹³C NMR(75 MHz, D₂O): δ 21.15 (isobutyl-2CH₃), 21.54 (NHCOCH₃ ), 28.09(isobutyl-CH), 33.42 (isobutyl-CH₂), 48.53 (C-5), 59.65 (C-4), 62.99(C-9), 67.98 (C-7), 69.63 (C-8), 75.29 (C-6), 102.06 (C-3), 122.01(triazole-C-5), 147.74 (triazole-C-4), 150.12 (C-2), 168.81 (COONa),173.28 (NHCOCH₃). LRMS [C₁₇H₂₅N₄NaO₇] (m/z): (+ve ion mode) 443.1[M+Na]⁺; HRMS (API) (m/z): [M+1]⁺ calcd for C₁₇H₂₆N₄NaO₇[M+1]⁺421.169370. found, 421.170091.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-(4-hydroxymethylphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE932-10)

¹H NMR (300 MHz, CDCl₃): ¹H NMR (300 MHz, Chloroform-d) δ 1.86 (s, 3H,NAc), 2.07 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.10 (s, 3H, OAc), 3.84 (s,3H, COOCH₃), 4.19 (dd, J=12.5, 6.6 Hz, 1H, H-9), 4.31 (m, 1H, H-5),4.60-4.74 (m, 3H, H-9′, CH₂), 4.80 (d, J=10.4 Hz, 1H, H-6), 5.40 (m, 1H,H-8), 5.54 (dd, J=5.8, 1.7 Hz, 1H, H-7), 5.93 (d, J=10.1 Hz, 1H, H-4),6.07 (d, J=2.2 Hz, 1H, H-3), 6.74 (brs, 1H, NH), 7.38 (d, J=7.9 Hz, 2H,Ph-H-3′, Ph-H-5′), 7.74 (d, J=7.7 Hz, 2H, Ph-H-2′, Ph-H-6′), 7.96 (s,1H, triazole-CH); ¹³H NMR (75 MHz, CDCl₃): δ 20.74, 20.85, 20.98 (3OCOCH₃ ), 22.90 (NHCOCH₃ ), 48.31 (C-5), 52.83 (COOCH₃), 58.49 (C-4),62.19 (C-9), 64.66 (CH₂), 67.72 (C-7), 70.93 (C-8), 76.78 (C-6), 107.12(C-3), 119.00 (triazole-C-5), 125.91 (Ph), 127.47 (Ph), 128.61 (Ph qcarbon), 141.59 (Ph q carbon), 146.01 (C-2), 147.65 (triazole-C-4),161.37 (COOCH₃), 170.13, 170.41, 170.92, 171.26 (NHCOCH₃, 3 OCOCH₃).LRMS [C₂₇H₃₂N₄O₁₁] (m/z): (+ve ion mode) 611.2 [M+Na]⁺.

Sodium 5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-(4-hydroxymethylphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-12)

¹H NMR (300 MHz, D₂O): δ 1.85 (s, 3H, NAc), 3.54-3.71 (m, 2H, H-9, H-7),3.86 (dd, J=11.9, 2.5 Hz, 1H, H-9′), 3.96 (ddd, J=9.3, 6.3, 2.4 Hz, 1H,H-8), 4.37 (m, 1H, H-5), 4.54 (d, J=10.9 Hz, 1H, H-6), 4.62 (s, 2H,CH₂), 5.50 (dd, J=9.6, 2.1 Hz, 1H, H-4), 5.83 (d, J=2.1 Hz, 1H, H-3),7.44 (d, J=8.2 Hz, 2H, Ph-H-3′, Ph-H-5′), 7.75 (d, J=8.2 Hz, 2H,Ph-H-2′, Ph-H-6′), 8.38 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, D₂O): δ21.70 (NHCOCH₃ ), 48.78 (C-5), 60.04 (C-4), 63.12 (C-9), 63.55 (CH₂),68.11 (C-7), 69.77 (C-8), 75.39 (C-6), 101.78 (C-3), 120.53(triazole-C-5), 125.90 (Ph), 128.06 (Ph), 128.77 (Ph q carbon), 140.80(Ph q carbon), 147.60 (C-2), 150.57 (triazole-C-4), 168.77 (COONa),173.66 (NHCOCH₃). LRMS [C₂₀H₂₃N₄NaO₈] (m/z): (+ve ion mode) 493.1[M+Na]⁺; HRMS (API) (m/z): [M+1]⁺ calcd for C₂₀H₂₄N₄NaO₈[M+1]⁺471.148635. found, 471.147973.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-methoxymethyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-13)

Purification by silica gel chromatography using ethylacetate: acetone(6:1) yielded (67 mg, 58%) of pure IE832-13. ¹H NMR (300 MHz, CDCl₃): δ1.81 (S, 3H, NAc), 2.05 (s, 6H, 2 OAc), 2.06 (s, 3H, OAc), 3.36 (s, 3H,OCH₃), 3.80 (s, 3H, COOCH₃), 4.17 (dd, J=12.5, 7.2 Hz, 1H, H-9), 4.29(m, 1H, H-5), 4.50 (s, 2H, OCH₂), 4.68-4.79 (m, 2H, H-9′, H-6), 5.40(ddd, J=7.4, 4.9, 2.5 Hz, 1H, H-8), 5.53 (dd, J=5.1, 1.8 Hz, 1H, H-7),5.78 (dd, J=10.0, 2.5 Hz, 1H, H-4), 6.00 (d, J=2.3 Hz, 1H, H-3), 7.05(d, J=9.1 Hz, 1H, NH), 7.64 (s, 1H, triazole-CH); ¹³C NMR (75 MHz,CDCl₃) δ 20.71, 20.79, 20.91 (3 OCOCH₃ ), 22.80 (NHCOCH₃ ), 48.39 (C-5),52.71 (COOCH₃ ), 58.16 (OCH₃), 58.38 (C-4), 62.21 (C-9), 65.68 (OCH₂),67.73 (C-7), 70.90 (C-8), 76.71 (C-6), 107.18 (C-3), 121.50(triazole-C-5), 145.24 (triazole-C-4), 145.92 (C-2), 161.27 (COOCH₃),170.06, 170.27, 170.81, 170.88 (NHCOCH₃, 3 OCOCH₃). LRMS[C₂₂H₃₀N₄O₁₁](m/z): (+ve ion mode) 549.1[M+Na]⁺. HRMS (API) (m/z):[M+Na]⁺ calcd for C₂₂H₃₀N₄NaO₁₁ [M+Na]⁺549.1803. found, 549.1805.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-methoxymethyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-17)

Yield=85%. ¹H NMR (300 MHz, D₂O): δ 1.84 (s, 3H, NAc), 3.31 (s, 3H,OCH₃), 3.52-3.71 (m, 2H, H-9 & H-7), 3.85 (dd, J=11.9, 2.6 Hz, 1H,H-9′), 3.95 (ddd, J=9.3, 6.2, 2.5 Hz, 1H, H-8), 4.33 (m, 1H, H-5), 4.51(dd, J=10.9, 1.2 Hz, 1H, H-6), 4.56 (s, 2H, OCH₂), 5.48 (dd, J=9.6, 2.3Hz, 1H, H-4), 5.80 (d, J=2.2 Hz, 1H, H-3), 8.08 (s, 1H, triazole-CH);¹³C NMR (75 MHz, D₂O): δ 21.65 (NHCOCH₃ ), 48.68 (C-5), 57.15 (OCH₃),59.94 (C-4), 63.06 (C-9), 64.22 (OCH₂), 68.05 (C-7), 69.71 (C-8), 75.34(C-6), 101.80 (C-3), 123.54 (triazole-C-5), 144.08 (C-2), 150.43(triazole-C-4), 168.75 (COONa), 173.57 (NHCOCH₃); LRMS [C₁₅H₂₁N₄NaO₈](m/z): (+ve ion mode) 432.1 [M+Na]⁺.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-(3-methoxyphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-18)

¹H NMR (300 MHz, CDCl₃): δ 1.84 (s, 3H, NAc), 2.05 (s, 3H, OAc), 2.06(s, 3H, OAc), 2.08 (s, 3H, OAc), 3.83 (s, 3H, COOCH₃), 3.84 (s, 3H,OCH₃), 4.20 (dd, J=12.5, 7.0 Hz, 1H, H-9), 4.32 (m, 1H, H-5), 4.71 (dd,J=12.5, 2.7 Hz, 1H, H9′), 4.83 (dd, J=10.5, 1.9 Hz, 1H, H-6), 5.43 (ddd,J=6.9, 5.4, 2.6 Hz, 1H, H-8), 5.57 (dd, J=5.4, 1.9 Hz, 1H, H-7), 5.87(dd, J=10.0, 2.5 Hz, 1H, H-4), 6.05 (d, J=2.4 Hz, 1H, H-3), 6.79-6.92(m, 2H, NH, Ph-H-4′), 7.23-7.38 (m, 3H, Ph-H-2′, Ph-H-5′, Ph-H-6′), 7.84(s, 1H, triazole-CH); ¹³C NMR (75 MHz, CDCl₃) δ 20.73, 20.79, 20.92 (3OCOCH₃ ), 22.98 (NHCOCH₃ ), 48.79 (C-5), 52.74 (COOCH₃ ), 55.35 (Ar—OCH₃), 58.04 (C-4), 62.18 (C-9), 67.74 (C-7), 70.78 (C-8), 76.63 (C-6),107.19 (C-3), 110.94 (Ph), 114.43 (Ph), 118.20 (Ph), 118.93(triazole-C-5), 129.97 (Ph), 131.17 (Ph q carbon), 146.02 (C2), 148.03(triazole-C-4), 160.02 (Ph q carbon), 161.31 (COOCH₃), 170.12, 170.24,170.81, 170.99 (NHCOCH₃, 3 OCOCH₃). LRMS [C₂₇H₃₂N₄O₁₁] (m/z): (+ve ionmode) 611.2 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-(3-methoxyphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-20)

¹H NMR (300 MHz, D₂O): δ 1.86 (s, 3H, NAc), 3.52-3.74 (m, 2H, H-9, H-7),3.77-3.92 (m, 4H, OCH₃, H-9′), 3.97 (m, 1H, H-8), 4.37 (m, 1H, H-5),4.54 (d, J=10.9 Hz, 1H, H-6), 5.49 (dd, J=9.6, 2.4 Hz, 1H, H-4), 5.84(s, 1H, H-3), 6.96 (m, 1H, Ph-H-4′), 7.23-7.48 (m, 3H, Ph-H-2′, Ph-H-5′,Ph-H-6′), 8.35 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, D₂O): δ 21.71(NHCOCH₃ ), 48.80 (C-5), 55.39 (OCH₃), 60.03 (C-4), 63.12 (C-9), 68.11(C-7), 69.77 (C-8), 75.39 (C-6), 101.73 (C-3), 110.89 (Ph), 114.54 (Ph),118.49 (Ph), 120.66 (triazole-C-5), 130.49 (Ph), 130.85 (Ph q carbon),147.51 (C2), 150.62 (triazole-C-4), 159.29 (Ph q carbon), 168.77(COONa), 173.65 (NHCOCH₃). LRMS [C₂₀H₂₃N₄NaO₈] (m/z): (+ve ion mode)493.0 [M+1]⁺; HRMS (API) (m/z): [M+1] calcd for C₂₀H₂₄N₄NaO₈[M+1]⁺471.148635. found, 471.148177.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-propyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-24)

¹H NMR (300 MHz, CDCl₃): δ 0.97 (t, J=7.3 Hz, 3H, propyl-CH₃), 1.73 (m,2H, propyl-2′-CH₂), 1.83 (s, 3H, NAc), 2.07 (s, 6H, 2 OAc), 2.10 (s, 3H,OAc), 2.77 (t, J=7.6 Hz, 2H, propyl-1′-CH₂), 3.83 (s, 3H, COOCH₃), 4.16(dd, J=12.5, 6.0 Hz, 1H, H-9), 4.38 (m, 1H, H-5), 4.58 (dd, J=12.6, 2.6Hz, 1H, H-9′), 4.85 (d, J=10.6 Hz, 1H, H-6), 5.42 (m, 1H, H-8), 5.56 (d,J=6.5 Hz, 1H, H-7), 6.01 (s, 1H, H-3), 6.11 (d, J=10.1 Hz, 1H, H-4),7.37 (brs, 1H, NH), 7.68 (s, 1H, triazole-CH); LRMS [C₂₃H₃₂N₄O₁₀] (m/z):(+ve ion mode) 547.2 [M+Na]⁺

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-propyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-26)

¹H NMR (300 MHz, D₂O): δ 0.92 (t, J=7.4 Hz, 3H, propyl-CH₃), 1.57-1.76(m, 2H, propyl-2′-CH₂), 1.93 (s, 3H, NAc), 2.71 (t, J=7.3 Hz, 2H,propyl-1′-CH₂), 3.62-3.77 (m, 2H, H-9, H-7), 3.94 (dd, J=11.9, 2.7 Hz,1H, H-9′), 4.04 (ddd, J=9.2, 6.3, 2.7 Hz, 1H, H-8), 4.41 (m, 1H, H-5),4.58 (dd, J=10.9, 1.3 Hz, 1H, H-6), 5.50 (dd, J=9.7, 2.3 Hz, 1H, H-4),5.87 (d, J=2.3 Hz, 1H, H-3), 7.89 (s, 1H, triazole-CH). LRMS[C₁₆H₂₃N₄NaO₇] (m/z): (+ve ion mode) 429.0 [M+Na]⁺; HRMS (API) (m/z):[M+Na]⁺ calcd for C₁₆H₂₃N₄Na₂O₇[M+1]⁺429.1357. found, 429.1361.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-(pyridin-3-yl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-25)

¹H NMR (300 MHz, CDCl₃): δ 1.82 (s, 3H, NAc), 2.06 (s, 6H, 2 OAc), 2.08(s, 3H, OAc), 3.82 (s, 3H, COOCH₃), 4.19 (dd, J=12.5, 7.2 Hz, 1H, H-9),4.40 (q, J=9.9 Hz, 1H, H-5), 4.64-4.88 (m, 2H, H-7, H-9′), 5.38 (m, 1H,H-8), 5.57 (dd, J=4.8, 1.9 Hz, 1H, H-7), 5.86 (dd, J=9.9, 2.4 Hz, 1H,H-4), 6.08 (d, J=2.4 Hz, 1H, H-3), 7.08 (d, J=9.1 Hz, 1H, NH), 7.36 (dd,J=7.9, 4.8 Hz, 1H, Pyr-H5′), 8.04 (s, 1H, triazole-CH), 8.14 (d, J=7.8Hz, 1H, Pyr-H4′), 8.54 (d, J=4.8 Hz, 1H, Pyr-H-6′), 8.96 (s, 1H,Pyr-H-2′); ¹³C NMR (75 MHz, CDCl₃) δ 20.69, 20.79, 20.94 (3 OCOCH₃ ),22.89 (NHCOCH₃ ), 48.35 (C-5), 52.81 (COOCH₃ ), 58.57 (C-4), 62.14(C-9), 67.77 (C-7), 71.17 (C-8), 76.85 (C-6), 106.88 (C-3), 119.07(triazole-C-5), 123.89 (Pyr), 126.33 (Pyr q carbon), 133.20 (Pyr),145.13 (triazole-C-4), 146.17 (C2), 146.88 (Pyr), 149.31 (Pyr), 161.25(COOCH₃), 170.16, 170.49, 170.80, 170.95 (NHCOCH₃, 3 OCOCH₃); LRMS[C₂₅H₂₉N₅O₁₀] (m/z): (+ve ion mode) 582.2 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-(pyridin-3-yl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-27)

¹H NMR (300 MHz, D₂O): δ 1.92 (s, 3H, NAc), 3.62-3.78 (m, 2H, H-9, H-7),3.93 (dd, J=11.9, 2.7 Hz, 1H, H-9′), 4.04 (ddd, J=9.3, 6.3, 2.6 Hz, 1H,H-8), 4.44 (m, 1H, H-5), 4.61 (dd, J=10.9, 1.3 Hz, 1H, H-6), 5.59 (dd,J=9.6, 2.3 Hz, 1H, H-4), 5.90 (d, J=2.3 Hz, 1H, H-3), 7.53 (m, 1H,Pyr-H-5′), 8.17 (d, J=8.0 Hz, 1H, Pyr-H-4′), 8.49-8.60 (m, 2H,triazole-CH, Phyr-H-6′), 8.87 (brs, 1H, Pyr-H-2′); ¹³C NMR (75 MHz,D₂O): δ 21.67 (NHCOCH₃ ), 48.77 (C-5), 60.17 (C-4), 63.08 (C-9), 68.06(C-7), 69.73 (C-8), 75.36 (C-6), 101.61 (C-3), 121.13 (triazole-C-5),134.31 (Pyr), 144.68 (C-2), 145.64 (Pyr), 148.42 (Pyr), 150.65(triazole-C-4), 168.72 (COONa), 173.65 (NHCOCH₃). LRMS[C₁₈H₂₀N₅NaO₇](m/z): (+ve ion mode) 463.7 [M+1]⁺; HRMS (API) (m/z):[M+1]⁺ calcd for C₁₈H₂₁N₅NaO₇ [M+1]⁺442.133319. found, 442.133358.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-(4-methoxyphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-29)

¹H NMR (300 MHz, CDCl₃): δ 1.88 (s, 3H, NAc), 2.06 (s, 3H, OAc), 2.10(s, 6H, 2 OAc), 3.83 (s, 3H, COOCH₃), 3.84 (s, 3H, OCH₃), 4.13-4.30 (m,2H, H-9, H-5), 4.66 (dd, J=12.5, 2.6 Hz, 1H, H-9′), 4.83 (dd, J=10.6,1.9 Hz, 1H, H-6), 5.41 (m, 1H, H-8), 5.52 (dd, J=5.8, 1.8 Hz, 1H, H-7),5.93 (dd, J=10.0, 2.5 Hz, 1H, H-4), 6.01-6.16 (m, 2H, H-3, NH), 6.94 (d,J=8.6 Hz, 2H, Ph-H-3′, Ph-H-5′), 7.61-7.83 (m, 3H, triazole-CH, Ph-H-2′,Ph-H-6′); ¹³C NMR (75 MHz, CDCl₃) δ 20.71, 20.78, 20.90 (3 OCOCH₃ ),22.90 (NHCOCH₃ ), 48.45 (C-5), 52.70 (COOCH₃ ), 55.31 (Ph-OCH₃ ), 58.17(C-4), 62.23 (C-9), 67.80 (C-7), 70.92 (C-8), 76.83 (C-6), 107.40 (C-3),114.32 (Ph), 117.88 (triazole-C-5), 122.57 (Ph q carbon), 127.13 (Ph),145.93 (C-2), 148.07 (triazole-C-4), 159.87 (Ph q carbon), 161.33(COOCH₃), 170.10, 170.27, 170.81, 170.99 (NHCOCH₃, 3 OCOCH₃). LRMS[C₂₇H₃₂N₄O₁₁] (m/z): (+ve ion mode) 611.3 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-(4-methoxyphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-31)

¹H NMR (300 MHz, D₂O): δ 1.92 (s, 3H, NAc), 3.63-3.77 (m, 2H, H-9, H-7),3.85 (s, 3H, OCH₃), 3.93 (dd, J=11.9, 2.7 Hz, 1H, H-9′), 4.03 (ddd,J=9.6, 6.3, 2.6 Hz, 1H, H-8), 4.42 (m, 1H, H-5), 4.59 (dd, J=10.9, 1.3Hz, 1H, H-6), 5.52 (dd, J=9.6, 2.3 Hz, 1H, H-4), 5.87 (d, J=2.2 Hz, 1H,H-3), 7.05 (d, J=9.0 Hz, 2H, Ph-H-3′, Ph-H-5′), 7.68 (d, J=9.0 Hz, 1H,Ph-H-2′, Ph-H-6′), 8.26 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, D₂O): δ21.65 (NHCOCH₃ ), 48.75 (C-5), 55.39 (OCH₃), 60.01 (C-4), 63.08 (C-9),68.07 (C-7), 69.72 (C-8), 75.35 (C-6), 101.82 (C-3), 114.60 (Ph), 119.78(triazole-C-5), 122.49 (Ph q carbon), 127.21 (Ph), 147.65 (C-2), 150.49(triazole-C-4), 159.23 (Ph q carbon), 168.78 (COONa), 173.62 (NHCOCH₃).LRMS [C₂₀H₂₃N₄NaO₈] (m/z): (+ve ion mode) 492.6 [M+1]⁺; HRMS (API)(m/z): [M+1]⁺ calcd for C₂₀H₂₄N₄NaO₈ [M+1]⁺471.148635. found,471.147452.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-(2-methoxyphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-30)

Purification by silica gel chromatography using ethylacetate: hexane(7:1) yielded (81 mg, 62%) of pure IE832-30. ¹H NMR (300 MHz, CDCl₃): δ1.84 (s, 3H, NAc), 2.05 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.08 (s, 3H,OAc), 3.83 (s, 3H, COOCH₃), 3.88 (s, 3H, OCH₃), 4.20 (dd, J=12.4, 7.0Hz, 1H, H-9), 4.37 (m, 1H, H-5), 4.71 (dd, J=12.4, 2.7 Hz, 1H, H-9′),4.89 (dd, J=10.5, 1.9 Hz, 1H, H-6), 5.45 (m, 1H, H-8), 5.59 (dd, J=5.4,1.9 Hz, 1H, H-7), 5.87 (dd, J=9.9, 2.5 Hz, 1H, H-4), 6.07 (d, J=2.4 Hz,1H, H-3), 6.91 (d, J=8.3 Hz, 1H, Ph-H-3′), 7.03 (m, 1H, Ph-H-5′),7.22-7.30 (m, 2H, Ph-H-4′, NH), 8.07 (s, 1H, triazole-CH), 8.17 (dd,J=7.8, 1.8 Hz, 1H, Ph-H-6′); ¹³C NMR (75 MHz, CDCl₃) b 20.71, 20.76,20.88 (3 OCOCH₃ ), 22.90 (NHCOCH₃ ), 48.84 (C-5), 52.64 (COOCH₃ ), 55.36(Ph-OCH₃ ), 57.83 (C-4), 62.24 (C-9), 67.85 (C-7), 70.79 (C-8), 76.75(C-6), 107.72 (C-3), 110.87 (Ph), 118.66 (Ph q carbon), 120.88 (Ph),122.32 (triazole-C-5), 127.37 (Ph), 129.24 (Ph), 143.40 (triazole-C4),145.82 (C-2), 155.68 (Ph q carbon), 161.44 (COOCH₃), 170.06, 170.19,170.75, 171.04 (NHCOCH₃, 3 OCOCH₃). LRMS [C₂₇H₃₂N₄O₁₁] (m/z): (+ve ionmode) 611.2 [M+Na]⁺; HRMS (API) (m/z): [M+Na]⁺ calcd for C₂₇H₃₂N₄NaO₁₁[M+Na]⁺611.195979. found, 611.196049.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-(2-methoxyphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-37)

Yield=91%. ¹H NMR (300 MHz, D₂O): δ 1.91 (s, 3H, NAc), 3.63-3.77 (m, 2H,H-9, H-7), 3.86-3.97 (m, 4H, H-9′, OCH₃), 4.04 (ddd, J=9.3, 6.3, 2.6 Hz,1H, H-8), 4.45 (m, 1H, H-5), 4.60 (dd, J=10.9, 1.2 Hz, 1H, H-6), 5.53(dd, J=9.6, 2.3 Hz, 1H, H-4), 5.90 (d, J=2.2 Hz, 1H, H-3), 7.08-7.14 (m,2H, Ph-H-3′, Ph-H-5′), 7.34-7.49 (m, 1H, Ph-H-4′), 7.92 (dd, J=8.0, 1.7Hz, H-6′), 8.32 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, D₂O): δ 21.67(NHCOCH₃ ), 48.59 (C-5), 55.38 (OCH₃), 59.81 (C-4), 63.09 (C-9), 68.11(C-7), 69.74 (C-8), 75.40 (C-6), 101.95 (C-3), 111.84 (Ph), 117.87 (Ph qcarbon), 121.00 (Ph), 123.12 (triazole-C-5), 127.27 (Ph), 130.05 (Ph),143.23 (triazole-C-4), 150.33 (C-2), 155.70 (Ph q carbon), 168.81(COONa), 173.59 (NHCOCH₃). LRMS [C₂₀H₂₃N₄NaO₈] (m/z): (+ve ion mode)493.2 [M+1]⁺.

Methyl7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(4-(2-methoxyphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE889-23)

¹H NMR (300 MHz, CDCl₃): δ 1.00-1.04 (m, 6H, isobutyryl-2CH₃), 2.08 (s,6H, 2 OAc), 2.10 (s, 3H, OAc), 2.25-2.34 (m, 1H, isobutyryl-CH), 3.84(s, 3H, COOCH₃), 3.88 (s, 3H, Ph-OCH₃), 4.20-4.28 (m, 2H, H-9, H-5),4.71 (dd, J=12.5, 2.6 Hz, 1H, H-9), 4.99 (d, J=10.5 Hz, 1H, H-6), 5.43(m, 1H, H-8), 5.55 (dd, J=5.4, 1.6 Hz, 1H, H-7), 6.03 (dd, J=10.1, 2.4Hz, 1H, H-4), 6.11 (d, J=2.3 Hz, 1H, H-3), 6.79 (d, J=8.3 Hz, 1H, NH),6.93 (d, J=8.3 Hz, 1H, Ph-H-3′), 7.05 (m, 1H, Ph-H-5′), 7.29 (m, 1H,Ph-H-4′), 8.04 (s, 1H, triazole-CH), 8.2 (d, J=7.9 Hz, 1H, Ph-H-6′); ¹³CNMR (75 MHz, CDCl₃): δ 18.86, 19.34, 20.80, 20.95 (3 OCOCH₃ ,isobutyryl-2CH₃), 35.42 (isobutyryl-CH), 49.31 (C-5), 52.68 (COOCH₃ ),55.29 (Ph-OCH₃ ), 57.12 (C-4), 62.19 (C-9), 67.74 (C-7), 70.75 (C-8),76.30 (C-6), 107.73 (C-3), 110.71 (Ph), 118.68 (Ph q carbon), 120.89(Ph), 122.62 (triazole-C-5), 127.51 (Ph), 129.21 (Ph), 143.30(triazole-C-5), 145.68 (C-2), 155.68 (Ph q carbon), 161.56 (COOCH₃),170.10, 170.20, 170.68 (3 OCOCH₃), 178.05 (isobutyryl-CO). LRMS[C₂₉H₃₆N₄O₁₁] (m/z): (+ve ion mode) 639.1 [M+Na]⁺; HRMS (API) (m/z):[M+Na]⁺ calcd for C₂₉H₃₆N₄NaO₁₁ [M+Na]⁺639.227279. found, 639.225897.

Sodium2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(4-(2-methoxyphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE889-34)

¹H NMR (300 MHz, D₂O): δ 0.90 (d, J=6.9 Hz, 3H, isobutyryl-CH₃), 0.97(d, J=6.9 Hz, 3H, isobutyryl-CH₃), 2.31 (m, 1H, isobutyryl-CH), 3.53 (d,J=9.4 Hz, 1H, H-7), 3.60 (dd, J=11.5, 5.4 Hz, 1H, H-9), 3.79 (dd,J=11.4, 3.0 Hz, 1H, H-9′), 3.85-3.91 (m, 4H, H-8, Ph-OCH₃), 4.39-4.55(m, 2H, H-5, H-6), 5.61 (dd, J=9.7, 2.2 Hz, 1H, H-4), 5.72 (d, J=2.2 Hz,1H, H-3), 6.89-7.10 (m, 2H, Ph-H-3′, Ph-H-5′), 7.27 (ddd, J=8.7, 7.4,1.7 Hz, 1H, H-4′), 8.02 (dd, J=7.7, 1.7 Hz, 1H, Ph-H-6′), 8.23 (s, 1H,triazole-CH); ¹³C NMR (75 MHz, D₂O): δ 18.38, 18.53 (isobutyryl-2CH₃),35.09 (isobutyryl-CH), 48.18 (C-5), 55.34 (OCH₃), 59.66 (C-4), 63.08(C-9), 68.18 (C-7), 69.76 (C-8), 75.44 (C-6), 102.15 (C-3), 111.80 (Ph),117.86 (Ph q carbon), 121.00 (Ph), 123.47 (triazole-C-5), 127.21 (Ph),130.05 (Ph), 143.12 (triazole-C-4), 150.16 (C-2), 155.70 (Ph q carbon),168.84 (COONa), 180.64 (NHCOCH₃); LRMS [C₂₂H₂₈N₄O₈] (m/z): (+ve ionmode) 499.1 [M+Na]⁺; HRMS (API) (m/z): [M+Na]⁺ calcd for C₂₂H₂₈N₄NaO₈[M+Na]⁺499.179935. found, 499.179943.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-phenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE889-45)

Purification by silica gel chromatography using ethylacetate: hexane(5:1) yielded (88 mg, 70%) of pure IE889-45. ¹H NMR (300 MHz, CDCl₃): δ1.79 (s, 3H, NAc), 2.04 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.06 (s, 3H,OAc), 3.81 (s, 3H, COOCH₃), 4.18 (dd, J=12.4, 7.2 Hz, 1H, H-9), 4.42 (m,1H, H-5), 4.72 (dd, J=12.6, 2.7 Hz, 1H, H-9′), 4.79 (dd, J=10.5, 1.6 Hz,1H, H-6), 5.42 (m, 1H, H-8), 5.58 (dd, J=5.1, 1.8 Hz, 1H, H-7), 5.83(dd, J=10.0, 2.4 Hz, 1H, H-4), 6.04 (d, J=2.3 Hz, 1H, H-3), 7.20-7.42(m, 4H, NH, Ph-H-3′, Ph-H-4′, Ph-H-5′), 7.72 (dd, J=8.2, 1.3 Hz, 2H,Ph-H-2′, Ph-H-6′), 7.88 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, CDCl₃) δ20.70, 20.80, 20.90 (3 OCOCH₃ ), 22.85 (NHCOCH₃ ), 48.26 (C-5), 52.71(COOCH₃ ), 58.41 (C-4), 62.26 (C-9), 67.80 (C-7), 70.97 (C-8), 76.95(C-6), 107.37 (C-3), 118.76 (triazole-C-5), 125.77 (Ph), 128.51 (Ph),128.91 (Ph), 129.85 (Ph, q carbon), 145.95 (C-2), 148.13 (triazole-C-4),161.32 (COOCH₃), 170.04, 170.30, 170.84, 170.99 (NHCOCH₃, 3 OCOCH₃).LRMS [C₂₆H₃₀N₄O₁₀] (m/z): (+ve ion mode) 581.0 [M+Na]⁺; HRMS (API)(m/z): [M+Na]⁺ calcd for C₂₆H₃₀N₄NaO₁₀ [M+Na]⁺ 581.185414. found,581.184724.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-phenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE889-52)

Yield=96%. ¹H NMR (300 MHz, D₂O): δ 1.87 (s, 3H, NAc), 3.64 (dd, J=12.1,6.4 Hz, 1H, H-9), 3.69 (dd, J=9.6, 1.4 Hz, 1H, H-7), 3.89 (dd, J=11.9,2.7 Hz, 1H, H-9′), 4.00 (ddd, J=9.3, 6.3, 2.7 Hz, 1H, H-8), 4.39 (m, 1H,H-5), 4.56 (dd, J=10.8, 1.4 Hz, 1H, H-6), 5.49 (dd, J=9.7, 2.3 Hz, 1H,H-4), 5.83 (d, J=2.2 Hz, 1H, H-3), 7.40 (m, 1H, Ph-H4′), 7.46 (dd,J=8.4, 6.9 Hz, 2H, Ph-H-3′, Ph-H-5′), 7.71 (d, J=7.1 Hz, 2H, Ph-H-2′,Ph-H-6′), 8.28 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, D₂O): δ 21.63(NHCOCH₃ ), 48.70 (C-5), 59.96 (C-4), 63.05 (C-9), 68.03 (C-7), 69.69(C-8), 75.31 (C-6), 101.75 (C-3), 120.41 (Ph), 125.61 (Ph), 128.77(triazole-C-5), 129.10 (Ph), 129.28 (Ph q carbon), 147.74(triazole-C-4), 150.48 (C-2), 168.75 (COONa), 173.58 (NHCOCH₃). LRMS[C₁₉H₂₁N₄NaO₇] (m/z): (+ve ion mode) 463.1 [M+Na]⁺; HRMS (API) (m/z):[M+1]⁺ calcd for C₁₉H₂₂N₄NaO₇ [M+H]⁺ 441.138070. found, 441.140189.

Methyl7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(4-methoxymethyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE927-57)

Purification by silica gel chromatography using ethylacetate: acetone(9:1) yielded (60 mg, 66%) of pure IE927-57. ¹H NMR (300 MHz, CDCl₃): δ0.97 (d, J=6.9 Hz, 3H, isobut-CH₃), 1.01 (d, J=6.8 Hz, 3H, isobut-CH₃),2.05 (s, 3H, OAc), 2.08 (s, 6H, 2 OAc), 2.24 (m, 1H, isobut-CH), 3.37(s, 3H, OCH₃), 3.81 (s, 3H, COOCH₃), 4.14-4.29 (m, 2H, H-9, H-5), 4.51(s, 2H, OCH₂), 4.68 (dd, J=12.5, 2.6 Hz, 1H, H-9′), 4.84 (dd, J=10.5,1.7 Hz, 1H, H-6), 5.38 (ddd, J=6.6, 5.5, 2.5 Hz, 1H, H-8), 5.48 (dd,J=5.5, 1.7 Hz, 1H, H-7), 5.91 (dd, J=10.0, 2.4 Hz, 1H, H-4), 6.02 (d,J=2.4 Hz, 1H, H-3), 6.44 (d, J=8.7 Hz, 1H, NH), 7.59 (s, 1H,triazole-CH); ¹³C NMR (75 MHz, CDCl₃): δ 18.81, 19.30, 20.74, 20.90 (3OCOCH₃ , isobut-2CH₃), 35.51 (isobut-CH), 48.79 (C-5), 52.69 (COOCH₃ ),57.59 (C-4), 58.38 (OCH₃), 62.08 (C-9), 65.73 (OCH₂), 67.66 (C-7), 70.79(C-8), 76.24 (C-6), 107.07 (C-3), 121.54 (triazole-C-5), 145.39(triazole-C-4), 145.82 (C-2), 161.30 (COOCH₃), 170.15, 170.23, 170.69 (3OCOCH₃), 177.87 (isobut-CO). LRMS [C₂₄H₃₄N₄O₁₁] (m/z): (+ve ion mode)577.2 [M+Na]⁺.

Sodium2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(4-methoxymethyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE927-60)

Yield=92%. ¹H NMR (300 MHz, D₂O): δ 0.98 (d, J=7.0 Hz, 3H, isobut-CH₃),1.03 (d, J=6.9 Hz, 3H, isobut-CH₃), 2.46 (m, 1H, isobut-CH), 3.39 (s,3H, OCH₃), 3.65-3.76 (m, 2H, H-9, H-7), 3.94 (dd, J=11.9, 2.7 Hz, 1H,H-9′), 4.04 (ddd, J=9.3, 6.3, 2.6 Hz, 1H, H-8), 4.49 (m, 1H, H-5),4.60-4.65 (m, 3H, H-6, OCH₂), 5.61 (dd, J=9.7, 2.3 Hz, 1H, H-4), 5.87(d, J=2.2 Hz, 1H, H-3), 8.18 (s, 1H, triazole-CH); ¹³C NMR (75 MHz,D₂O): δ 18.43 (isobut-CH₃), 18.64 (isobut-CH₃), 35.10 (isobut-CH), 48.19(C-5), 57.24 (OCH₃), 59.86 (C-4), 63.07 (C-9), 64.24 (OCH₂), 68.13(C-7), 69.82 (C-8), 75.43 (C-6), 102.02 (C-3), 123.65 (triazole-C-5),144.07 (triazole-C-4), 150.30 (C-2), 168.81 (COONa), 180.66 (isobut-CO).LRMS [C₁₇H₂₅N₄NaO₈] (m/z): (+ve ion mode) 459.0 [M+Na]⁺; HRMS (API)(m/z): [M+Na]⁺ calcd for C₁₇H₂₅N₄Na₂O₈[M+Na]⁺459.1462. found, 459.1458.

Methyl7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-isobutyl-[1,2,3,]triazol-1-yl)-5-isobutyramido-D-glycero-D-galacto-non-2-enonate(IE927-58)

¹H NMR (300 MHz, CDCl₃): δ 0.90 (d, J=6.6 Hz, 6H, isobutyl-2CH₃), 0.96(d, J=6.9 Hz, 3H, isobutyryl-CH₃), 1.00 (d, J=6.8 Hz, 3H,isobutyryl-CH₃), 1.90 (m, 1H, isobutyl-CH), 2.05 (s, 3H, OAc), 2.08 (s,6H, 2 OAc), 2.22 (m, 1H, isobutyryl-CH), 2.53 (d, J=7.0 Hz, 2H,isobutyl-CH₂), 3.82 (s, 3H, COOCH₃), 4.13-4.36 (m, 2H, H-9, H-5), 4.70(dd, J=12.5, 2.6 Hz, 1H, H-9′), 4.81 (dd, J=10.5, 1.7 Hz, 1H, H-6), 5.38(ddd, J=6.6, 5.4, 2.5 Hz, 1H, H-8), 5.49 (dd, J=5.5, 1.7 Hz, 1H, H-7),5.87 (dd, J=10.1, 2.4 Hz, 1H, H-4), 6.02 (d, J=2.4 Hz, 1H, H-3), 6.41(d, J=8.8 Hz, 1H, NH), 7.32 (s, 1H, triazole-CH); ¹³C NMR (75 MHz,CDCl₃): δ 18.83, 19.29, 20.76, 20.92 (3 OCOCH₃ +isobutyryl-2CH₃), 22.23(isobutyl-2CH₃), 28.70 (isobutyl-CH), 34.68 (isobutyl-CH₂), 35.55(isobutyryl-CH), 48.48 (C-5), 52.68 (COOCH₃ ), 57.47 (C-4), 62.11 (C-9),67.69 (C-7), 70.89 (C-8), 76.44 (C-6), 107.53 (C-3), 120.36(triazole-C-5), 145.56 (C-2), 147.53 (triazole-C-4), 161.40 (COOCH₃),170.15, 170.24, 170.70 (3 OCOCH₃), 177.74 (isobutyryl-CO). LRMS[C₂₆H₃₈N₄O₁₀] (m/z): (+ve ion mode) 589.3 [M+Na]⁺.

Sodium2,6-anhydro-3,4,5-trideoxy-4-(4-isobutyl-[1,2,3,]triazol-1-yl)-5-isobutyramido-D-glycero-D-galacto-non-2-enonate

¹H NMR (300 MHz, D₂O): δ 0.90 (d, J=6.6 Hz, 6H, isobutyl-2CH₃), 0.97 (d,J=6.9 Hz, 3H, isobutyryl-CH₃), 1.02 (d, J=6.9 Hz, 3H, isobutyryl-CH₃),1.92 (m, 1H, isobutyl-CH), 2.43 (m, 1H, isobutyryl-CH), 2.60 (d, J=6.9Hz, 2H, isobutyl-CH₂), 3.62-3.74 (m, 2H, H-9, H-7), 3.93 (dd, J=11.9,2.7 Hz, 1H, H-9′), 4.02 (ddd, J=9.3, 6.3, 2.6 Hz, 1H, H-8), 4.47 (m, 1H,H-5), 4.56 (d, J=11.1 Hz, 1H, H-6), 5.53 (dd, J=9.7, 2.2 Hz, 1H, H-4),5.84 (d, J=2.2 Hz, 1H, H-3), 7.88 (s, 1H, triazole-CH); ¹³C NMR (75 MHz,D₂O): δ 18.45 (isobutyryl-CH₃), 18.61 (isobutyryl-CH₃), 21.31(isobutyl-2CH₃), 28.17 (isobutyl-CH), 33.54 (isobutyl-CH₂), 35.12(isobutyryl-CH), 48.09 (C-5), 59.53 (C-4), 63.07 (C-9), 68.14 (C-7),69.78 (C-8), 75.47 (C-6), 102.30 (C-3), 121.99 (triazole-C-5), 147.82(triazole-C-4), 150.05 (C-2), 168.88 (COONa), 180.46 (isobutyryl-CO).LRMS [C₁₉H₂₉N₄NaO₇] (m/z): (+ve ion mode) 471.2 [M+Na]⁺, 449.2; HRMS(API) (m/z): [M+Na]⁺ calcd for C₁₉H₂₉N₄Na₂O₇[M+Na]⁺471.1826. found,471.1823.

Methyl7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(4-phenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE984-4)

Purification by silica gel chromatography using ethylacetate: hexane(4:1) yielded (72 mg, 74%) of pure IE984-4. ¹H NMR (300 MHz, CDCl₃): δ0.95 (d, J=6.8 Hz, 3H, isobut-CH₃), 0.99 (d, J=6.9 Hz, 3H, isobut-CH₃),2.06 (s, 3H, OAc), 2.09 (s, 6H, 2 OAc), 2.20-2.27 (m, 1H, isobut-CH),3.83 (s, 3H, COOCH₃), 4.16-4.39 (m, 2H, H-9, H-5), 4.70 (dd, J=12.5, 2.6Hz, 1H, H-9′), 4.88 (dd, J=10.5, 1.7 Hz, 1H, H-6), 5.40 (m, 1H, H-8),5.52 (dd, J=5.4, 1.7 Hz, 1H, H-7), 5.99 (dd, J=10.0, 2.4 Hz, 1H, H-4),6.08 (d, J=2.4 Hz, 1H, H-3), 6.51 (d, J=8.7 Hz, 1H, NH), 7.26-7.43 (m,3H, Ph-H-3′, Ph-H-4′, Ph-H-5′), 7.74 (d, J=7.2 Hz, 2H, Ph-H-2′,Ph-H-5′), 7.81 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, CDCl₃): δ 18.83,19.31, 20.76, 20.92 (3 OCOCH₃ , isobut-2CH₃), 35.55 (isobut-CH), 48.74(C-5), 52.72 (COOCH₃ ), 57.67 (C-4), 62.12 (C-9), 67.72 (C-7), 70.84(C-8), 76.39 (C-6), 107.25 (C-3), 118.84 (triazole-C-5), 125.83 (Ph),128.47 (Ph), 128.89 (Ph), 129.97 (Ph q carbon), 145.81 (C-2), 148.19(triazole-C-4), 161.35 (COOCH₃), 170.18, 170.26, 170.71 (3 OCOCH₃),178.00 (isobut-CO). LRMS [C₂₈H₃₄N₄O₁₀] (m/z): (+ve ion mode) 608.9[M+Na]⁺.

Sodium2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(4-phenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE984-5)

Yield=89%. ¹H NMR (300 MHz, D₂O): δ 0.94 (d, J=6.9 Hz, 3H, isobut-CH₃),0.99 (d, J=6.9 Hz, 3H, isobut-CH₃), 2.43 (m, 1H, isobut-CH), 3.60-3.76(m, 2H, H-9, H-7), 3.93 (dd, J=12.0, 2.7 Hz, 1H, H-9′), 4.04 (ddd,J=9.2, 6.3, 2.6 Hz, 1H, H-8), 4.51 (m, 1H, H-5), 4.62 (d, J=11.0 Hz, 1H,H6), 5.58 (dd, J=9.7, 2.3 Hz, 1H, H-4), 5.88 (d, J=2.2 Hz, 1H, H-3),7.42-7.54 (m, 3H, Ph-H-3′, Ph-H-4′, Ph-H-5′), 7.78 (d, J=7.1 Hz, 2H,Ph-H-2′, Ph-H-6′), 8.36 (s, 1H, triazole-CH); ¹³C NMR (75 MHz, D₂O): δ18.38 (isobut-CH₃), 18.65 (isobut-CH₃), 35.10 (isobut-CH), 48.23 (C-5),59.91 (C-4), 63.07 (C-9), 68.15 (C-7), 69.76 (C-8), 75.41 (C-6), 101.96(C-3), 120.66 (Ph), 125.67 (Ph), 128.81 (triazole-C-5), 129.16 (Ph),129.36 (Ph q carbon), 147.71 (triazole-C-4), 150.32 (C-2), 168.80(COONa), 180.67 (isobut-CO). LRMS [C₂₁H₂₅N₄NaO₇] (m/z): (+ve ion mode)491.2 [M+Na]⁺; HRMS (API) (m/z): [M+Na]⁺ calcd forC₂₁H₂₅N₄Na₂O₇[M+Na]⁺491.1513. found, 491.1515.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-(4-(2-chlorophenyl)-[1,2,3,]triazol-1-yl)-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(IE1172-70)

Purification by silica gel chromatography using acetone: hexane (3:2)yielded (76%) of pure IE1172-70. ¹H NMR (400 MHz, CDCl₃): δ 1.81 (s, 3H,NAc), 2.05 (s, 6H, 2 OAc), 2.07 (s, 3H, OAc), 3.82 (s, 3H, COOCH₃), 4.20(dd, J=12.5, 7.0 Hz, 1H, H-9), 4.30 (q, J=9.8 Hz, 1H, H-5), 4.71 (dd,J=12.5, 2.7 Hz, 1H, H-9′), 4.86 (dd, J=10.7, 1.9 Hz, 1H, H-6), 5.42(ddd, J=6.8, 5.4, 2.6 Hz, 1H, H-8), 5.55 (dd, J=5.3, 1.9 Hz, 1H, H-7),5.91 (dd, J=10.0, 2.4 Hz, 1H, H-4), 6.09 (d, J=2.3 Hz, 1H, H-3), 6.93(d, J=8.8 Hz, 1H, NH), 7.26 (td, J=7.8, 1.8 Hz, 1H, Ph-H), 7.34 (td,J=7.6, 1.4 Hz, 1H, Ph-H), 7.41 (dd, J=8.0, 1.3 Hz, 1H, Ph-H), 8.05 (dd,J=7.9, 1.7 Hz, 1H, Ph-H), 8.19 (s, 1H, triazole-CH); ¹³C NMR (101 MHz,CDCl₃) δ 20.75, 20.80, 20.93 (3 OCOCH₃ ), 22.94 (NHCOCH₃ ), 49.00 (C-5),52.74 (COOCH₃ ), 57.92 (C-4), 62.18 (C-9), 67.81 (C-7), 70.86 (C-8),76.48 (C-6), 107.11 (C-3), 122.63 (triazole-C-5), 127.22 (Ph), 128.62(Ph, q carbon), 129.44 (Ph), 129.80 (Ph), 130.28 (Ph), 131.42 (Ph, qcarbon), 144.31 (triazole-C-4), 145.96 (C-2), 161.34 (COOCH₃), 170.20,170.28, 170.79, 171.01 (NHCOCH₃, 3 OCOCH₃); LRMS [C₂₆H₂₉ClN₄O₁₀] (m/z):(+ve ion mode) 615.1 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-4-(4-(2-chlorophenyl)-[1,2,3,]triazol-1-yl)-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(IE1172-78)

Yield=89%. ¹H NMR (400 MHz, D₂O): δ 1.93 (s, 3H, NAc), 3.68 (dd, J=12.0,6.4 Hz, 1H, H-9), 3.73 (dd, J=9.7, 1.3 Hz, 1H, H-7), 3.93 (dd, J=12.0,2.7 Hz, 1H, H-9′), 4.03 (ddd, J=9.3, 6.3, 2.6 Hz, 1H, H-8), 4.48 (t,J=10.3 Hz, 1H, H-5), 4.61 (dd, J=11.1, 1.3 Hz, 1H, H-6), 5.58 (dd,J=9.7, 2.3 Hz, 1H, H-4), 5.93 (d, J=2.2 Hz, 1H, H-3), 7.37-7.51 (m, 2H,2Ph-H), 7.58 (dd, J=7.5, 1.9 Hz, 1H, Ph-H), 7.81 (dd, J=7.0, 2.5 Hz, 1H,Ph-H), 8.49 (s, 1H, triazole-CH); ¹³C NMR (101 MHz, D₂O): δ 21.70(NHCOCH₃ ), 48.67 (C-5), 60.10 (C-4), 63.09 (C-9), 68.10 (C-7), 69.74(C-8), 75.39 (C-6), 101.87 (C-3), 123.78 (triazole-C-5), 127.36 (Ph),128.03 (Ph q carbon), 129.94 (Ph), 130.10 (Ph), 130.19 (Ph), 131.50 (Phq carbon), 144.37 (triazole-C-4), 150.41 (C-2), 168.78 (COONa), 173.56(NHCOCH₃); LRMS [C₁₉H₂₀ClN₄NaO₇] (m/z): (+ve ion mode) 497.1 [M+Na]⁺.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-(2-methylphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1172-72)

Purification by silica gel chromatography using acetone: hexane (4:3)yielded (81%) of pure IE1172-72. ¹H NMR (400 MHz, CDCl₃): δ 1.76 (s, 3H,NAc), 2.04 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.33 (s,3H, Ph-CH₃), 3.82 (s, 3H, COOCH₃), 4.19 (dd, J=12.5, 7.1 Hz, 1H, H-9),4.39 (q, J=9.9 Hz, 1H, H-5), 4.71 (dd, J=12.4, 2.7 Hz, 1H, H-9′), 4.82(dd, J=10.4, 2.0 Hz, 1H, H-6), 5.41 (ddd, J=7.6, 5.1, 2.7 Hz, 1H, H-8),5.57 (dd, J=5.2, 1.9 Hz, 1H, H-7), 5.86 (dd, J=10.2, 2.4 Hz, 1H, H-4),6.07 (d, J=2.3 Hz, 1H, H-3), 7.16-7.30 (m, 4H, NH, 3PH—H), 7.60 (m, 1H,Ph-H), 7.70 (s, 1H, triazole-CH); ¹³C NMR (101 MHz, CDCl₃) δ 20.72,20.81, 20.93 (3 OCOCH₃ ), 21.09 (Ph-CH₃), 22.82 (NHCOCH₃ ), 48.63 (C-5),52.73 (COOCH₃ ), 58.03 (C-4), 62.21 (C-9), 67.76 (C-7), 70.93 (C-8),76.72 (C-6), 107.38 (C-3), 121.28 (triazole-C-5), 126.16 (Ph), 128.58(Ph), 128.94 (Ph), 129.23 (Ph, q carbon), 130.90 (Ph), 135.64 (Ph, qcarbon), 145.85 (C-2), 147.15 (triazole-C-4), 161.37 (COOCH₃), 170.07,170.30, 170.80, 171.06 (NHCOCH₃, 3 OCOCH₃); LRMS [C₂₇H₃₂N₄O₁₀] (m/z):(+ve ion mode) 595.2 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-(2-methylphenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1172-82)

Yield=85%. ¹H NMR (400 MHz, D₂O): δ 1.93 (s, 3H, NAc), 2.36 (s, 3H,Ph-CH₃), 3.68 (dd, J=11.9, 6.3 Hz, 1H, H-9), 3.74 (d, J=9.7 Hz, 1H,H-7), 3.93 (dd, J=12.0, 2.7 Hz, 1H, H-9′), 4.03 (ddd, J=9.3, 6.3, 2.6Hz, 1H, H-8), 4.48 (t, J=10.3 Hz, 1H, H-5), 4.60 (dd, J=10.8, 1.3 Hz,1H, H-6), 5.58 (dd, J=9.7, 2.3 Hz, 1H, H-4), 5.92 (d, J=2.3 Hz, 1H,H-3), 7.29-7.46 (m, 3H, 3Ph-H), 7.58 (d, J=7.4 Hz, 1H, Ph-H), 8.19 (s,1H, triazole-CH); ¹³C NMR (101 MHz, D₂O): δ 19.77 (Ph-CH₃ ), 21.67(NHCOCH₃ ), 48.69 (C-5), 60.06 (C-4), 63.09 (C-9), 68.10 (C-7), 69.74(C-8), 75.39 (C-6), 101.92 (C-3), 122.60 (triazole-C-5), 126.17 (Ph),128.94 (Ph q carbon), 129.01 (Ph), 129.03 (Ph), 130.76 (Ph), 136.45 (Phq carbon), 146.87 (triazole-C-4), 150.42 (C-2), 168.80 (COONa), 173.52(NHCOCH₃); LRMS [C₂₀H₂₃N₄NaO₇] (m/z): (+ve ion mode) 477.2 [M+Na]⁺.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-(2-(trifluoromethyl)phenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1172-74)

Purification by silica gel chromatography using acetone: hexane (3:2)yielded (90%) of pure IE1172-74. ¹H NMR (400 MHz, CDCl₃): δ 1.75 (s, 3H,NAc), 2.04 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.08 (s, 3H, OAc), 3.83 (s,3H, COOCH₃), 4.14-4.35 (m, 2H, H-9, H-5), 4.70 (dd, J=12.4, 2.6 Hz, 1H,H-9′), 4.92 (dd, J=10.6, 1.8 Hz, 1H, H-6), 5.42 (ddd, J=6.8, 5.4, 2.6Hz, 1H, H-8), 5.54 (dd, J=5.5, 1.8 Hz, 1H, H-7), 5.97 (dd, J=10.1, 2.4Hz, 1H, H-4), 6.08 (d, J=2.3 Hz, 1H, H-3), 6.99 (d, J=8.5 Hz, 1H, NH),7.48 (t, J=7.7 Hz, 1H, Ph-H), 7.61 (td, J=7.7, 1.4 Hz, 1H, Ph-H), 7.72(dd, J=8.1, 1.4 Hz, 1H, Ph-H), 7.75-7.84 (m, 2H, Ph-H, triazole-CH); ¹³CNMR (101 MHz, CDCl₃) δ 20.76, 20.79, 20.92 (3 OCOCH₃ ), 22.73 (NHCOCH₃), 49.38 (C-5), 52.74 (COOCH₃ ), 57.48 (C-4), 62.16 (C-9), 67.80 (C-7),70.79 (C-8), 76.25 (C-6), 107.09 (C-3), 122.58 (Ph, q carbon), 122.82(q, J=5.1, 4.6 Hz, triazole-C-5), 125.29 (Ph, q carbon), 126.22 (q,J=5.5 Hz, Ph), 127.61 (q, J=30.4 Hz, CF₃), 128.71 (Ph), 131.79 (Ph),132.09 (Ph), 144.40 (triazole-C-4), 145.84 (C-2), 161.35 (COOCH₃),170.20, 170.26, 170.75, 171.19 (NHCOCH₃, 3 OCOCH₃); LRMS [O₂₇H₂₉F₃N₄O₁₀](m/z): (+ve ion mode) 649.1 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-(2-(trifluoromethyl)phenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1172-83)

Yield=77%. ¹H NMR (400 MHz, D₂O): δ 1.94 (s, 3H, NAc), 3.68 (dd, J=11.8,6.3 Hz, 1H, H-9), 3.73 (d, J=9.6 Hz, 1H, H-7), 3.93 (dd, J=11.9, 2.7 Hz,1H, H-9′), 4.03 (ddd, J=9.3, 6.2, 2.6 Hz, 1H, H-8), 4.51 (t, J=10.2 Hz,1H, H-5), 4.59 (d, J=10.9 Hz, 1H, H-6), 5.61 (dd, J=9.7, 2.3 Hz, 1H,H-4), 5.93 (d, J=2.2 Hz, 1H, H-3), 7.61-7.70 (m, 2H, 2Ph-H), 7.75 (t,J=7.6 Hz, 1H, Ph-H), 7.90 (d, J=7.8 Hz, 1H, Ph-H), 8.23 (s, 1H,triazole-CH); ¹³C NMR (101 MHz, D₂O): δ 21.61 (NHCOCH₃ ), 48.62 (C-5),59.96 (C-4), 63.09 (C-9), 68.10 (C-7), 69.73 (C-8), 75.48 (C-6), 101.96(C-3), 122.49 (Ph q carbon), 123.77 (q, J=3.0 Hz, triazole-C-5), 125.20(Ph q carbon), 126.32 (q, J=5.4 Hz, Ph), 127.39-128.51 (m, CF₃), 129.38(Ph), 131.99 (Ph), 132.27 (Ph), 144.83 (triazole-C-4), 150.33 (C-2),168.75 (COONa), 173.52 (NHCOCH₃); LRMS [C₂₀H₂₀F3N₄NaO₇](m/z): (+ve ionmode) 531.2 [M+Na]⁺.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(5-methoxymethyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1172-79)

Purification by silica gel chromatography using ethylacetate: acetone(6:1) yielded (68%) of pure IE1172-79. ¹H NMR (400 MHz, CDCl₃): δ 1.87(s, 3H, NAc), 2.01 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.07 (s, 3H, OAc),3.34 (s, 3H, CH₂OCH₃ ), 3.79 (s, 3H, COOCH₃), 4.15-4.27 (m, 2H, H-5,H-9), 4.34-4.50 (m, 2H, CH₂O), 4.60 (dd, J=12.5, 2.6 Hz, 1H, H-9′), 5.09(dd, J=10.7, 1.8 Hz, 1H, H-6), 5.42 (td, J=6.2, 2.6 Hz, 1H, H-8), 5.49(dd, J=6.3, 1.8 Hz, 1H, H-7), 5.91 (d, J=2.4 Hz, 1H, H-3), 6.05 (dd,J=9.9, 2.5 Hz, 1H, H-4), 7.26 (d, J=7.8 Hz, 1H, NH), 7.46 (s, 1H,triazole-CH); ¹³C NMR (101 MHz, CDCl₃) δ 20.71, 20.76, 20.92 (3 OCOCH₃), 23.04 (NHCOCH₃ ), 50.03 (C-5), 52.60 (COOCH₃ ), 55.20 (C-4), 58.59(CH₂OCH₃ ), 61.94 (CH₂ OCH₃), 62.10 (C-9), 67.74 (C-7), 70.25 (C-8),75.54 (C-6), 107.87 (C-3), 133.48 (triazole-C-4), 134.46 (triazole-C-5),145.50 (C-2), 161.55 (COOCH₃), 169.96, 170.21, 170.66, 171.54 (NHCOCH₃,3 OCOCH₃); LRMS [C₂₂H₃₀N₄O₁₁] (m/z): (+ve ion mode) 549.3 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(5-methoxymethyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1172-87)

Yield=83%. ¹H NMR (400 MHz, D₂O): δ 1.89 (s, 3H, NAc), 3.40 (s, 3H,OCH₃), 3.65-3.74 (m, 2H, H-9, H-7), 3.93 (dd, J=12.0, 2.7 Hz, 1H, H-9′),4.03 (ddd, J=9.3, 6.3, 2.6 Hz, 1H, H-8), 4.57 (d, J=10.9 Hz, 1H, H-6),4.61-4.73 (m, 3H, H-5, OCH₂), 5.62 (dd, J=9.6, 2.4 Hz, 1H, H-4), 5.85(d, J=2.4 Hz, 1H, H-3), 7.81 (s, 1H, triazole-CH); ¹³C NMR (101 MHz,D₂O): δ 21.73 (NHCOCH₃ ), 47.97 (C-5), 57.87 (OCH₃), 58.92 (C-4), 61.34(OCH₂), 63.08 (C-9), 68.08 (C-7), 69.70 (C-8), 75.34 (C-6), 102.36(C-3), 134.66 (triazole-C-5), 134.73 (triazole-C-4), 150.02 (C-2),168.75 (COONa), 173.61 (NHCOCH₃); LRMS [C₁₅H₂₁N₄NaO₈](m/z): (+ve ionmode) 431.2 [M+Na]⁺.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(5-phenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1172-39)

Purification by silica gel chromatography using Hexane: acetone (4:3)yielded (82%) of pure IE1172-39. ¹H NMR (400 MHz, CDCl₃): δ 1.74 (s, 3H,NAc), 2.02 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.08 (s, 3H, OAc), 3.80 (s,3H, COOCH₃), 4.04 (td, J=10.1, 7.3 Hz, 1H, H-5), 4.21 (m, 1H, H-9), 4.51(m, 1H, H-9′), 5.05 (d, J=10.5 Hz, 1H, H-6), 5.31-5.46 (m, 2H, H-7,H-8), 5.98 (d, J=2.5 Hz, 1H, H-3), 6.09 (dd, J=9.7, 2.5 Hz, 1H, H-4),6.75 (d, J=7.4 Hz, 1H, NH), 7.30-7.32 (m, 2H, 2Ph-H), 7.47-7.53 (m, 3H,3Ph-H), 7.58 (s, 1H, triazole-CH); ¹³C NMR (101 MHz, CDCl₃) δ 20.73,20.79, 20.89 (3 OCOCH₃ ), 23.03 (NHCOCH₃ ), 50.85 (C-5), 52.58 (COOCH₃), 54.24 (C-4), 62.01 (C-9), 67.63 (C-7), 69.87 (C-8), 74.78 (C-6),108.02 (C-3), 126.01 (Ph q carbon), 129.07 (Ph), 129.23 (Ph), 130.07(Ph), 132.70 (triazole-C-4), 139.35 (triazole-C-5), 145.49 (C-2), 161.49(COOCH₃), 169.79, 170.40, 170.58, 171.03 (NHCOCH₃, 3 OCOCH₃); LRMS[C₂₆H₃₀N₄O₁₀] (m/z): (+ve ion mode) 581.1 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(5-phenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1172-45)

Yield=88%. ¹H NMR (400 MHz, D₂O): δ 1.72 (s, 3H, NAc), 3.55 (d, J=9.7Hz, 1H, H-7), 3.62 (dd, J=12.0, 6.3 Hz, 1H, H-9), 3.87 (dd, J=11.9, 2.7Hz, 1H, H-9′), 3.95 (ddd, J=9.3, 6.2, 2.7 Hz, 1H, H-8), 4.33-4.51 (m,2H, H-5, H-6), 5.63 (dd, J=9.3, 2.2 Hz, 1H, H-4), 5.98 (d, J=2.3 Hz, 1H,H-3), 7.46-7.56 (m, 2H, 2Ph-H), 7.57-7.61 (m, 3H, 3Ph-H), 7.85 (s, 1H,triazole-CH); ¹³C NMR (101 MHz, D₂O): δ 21.88 (NHCOCH₃ ), 48.83 (C-5),57.52 (C-4), 63.00 (C-9), 67.95 (C-7), 69.64 (C-8), 75.01 (C-6), 103.14(C-3), 125.71 (Ph q carbon), 129.13 (Ph), 129.28 (Ph), 130.10 (Ph),133.02 (triazole-C-4), 139.88 (triazole-C-5), 149.90 (C-2), 168.80(COONa), 173.30 (NHCOCH₃); LRMS [C₁₉H₂₁N₄NaO₇](m/z): (+ve ion mode)462.6 [M+Na]⁺.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4,5-diphenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1172-90)

Purification by silica gel chromatography using Hexane: acetone (3:2)yielded (79%) of pure IE1172-90. ¹H NMR (400 MHz, CDCl₃): δ 1.82 (s, 3H,NAc), 2.03 (s, 6H, 2 OAc), 2.10 (s, 3H, OAc), 3.78 (s, 3H, COOCH₃),4.17-4.27 (m, 2H, H-5, H-9), 4.52 (dd, J=12.5, 2.6 Hz, 1H, H-9′), 5.07(d, J=10.4 Hz, 1H, H-6), 5.40 (td, J=6.3, 2.5 Hz, 1H, H-8), 5.45 (dd,J=6.8, 1.7 Hz, 1H, H-7), 5.79-5.93 (m, 2H, H-3, H-4), 7.13-7.27 (m, 6H,NH, 5Ph-H), 7.28-7.34 (m, 2H, 2Ph-H), 7.38-7.48 (m, 3H, 3Ph-H); ¹³C NMR(101 MHz, CDCl₃) δ 20.75, 20.76, 20.89 (3 OCOCH₃ ), 23.33 (NHCOCH₃ ),50.56 (C-5), 52.54 (COOCH₃ ), 54.97 (C-4), 62.09 (C-9), 67.54 (C-7),69.93 (C-8), 75.13 (C-6), 108.41 (C-3), 126.48 (Ph q carbon), 127.15(Ph), 128.09 (Ph), 128.44 (Ph), 129.31 (Ph), 130.11 (Ph), 130.14 (Ph),130.18 (Ph q carbon), 135.11 (triazole-C), 144.35 (triazole-C), 145.49(C-2), 161.50 (COOCH₃), 169.80, 170.27, 170.63, 171.37 (NHCOCH₃, 3OCOCH₃); LRMS [C₃₂H₃₄N₄O₁₀] (m/z): (+ve ion mode) 657.3 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4,5-diphenyl-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1172-102)

Yield=84%. ¹H NMR (400 MHz, D₂O): δ 1.83 (s, 3H, NAc), 3.58 (d, J=9.7Hz, 1H, H-7), 3.63 (dd, J=12.0, 6.2 Hz, 1H, H-9), 3.88 (dd, J=12.0, 2.7Hz, 1H, H-9′), 3.94 (ddd, J=9.4, 6.2, 2.7 Hz, 1H, H-8), 4.37 (d, J=11.0Hz, 1H, H-6), 4.59 (dd, J=11.2, 9.2 Hz, 1H, H-5), 5.48 (d, J=9.7 Hz, 1H,H-4), 5.90 (s, 1H, H-3), 7.36-7.59 (m, 10H, Ph-H); ¹³C NMR (101 MHz,D₂O): δ 21.94 (NHCOCH₃ ), 48.33 (C-5), 58.44 (C-4), 63.01 (C-9), 67.98(C-7), 69.61 (C-8), 74.96 (C-6), 103.15 (C-3), 126.05 (Ph q carbon),127.31 (Ph), 128.55 (Ph), 128.80 (Ph), 129.21 (Ph), 129.63 (Ph qcarbon), 130.25 (Ph), 130.42 (Ph), 135.79 (triazole-C), 144.67(triazole-C), 149.71 (C-2), 168.67 (COONa), 173.41 (NHCOCH₃); LRMS[C₂₅H₂₅N₄NaO₇] (m/z): (+ve ion mode) 539.2 [M+Na]⁺.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-(4-(2-bromophenyl)-[1,2,3,]triazol-1-yl)-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(IE1257-75)

Purification by silica gel chromatography using hexane:acetone (3:2)yielded (88%) of pure IE1257-75. ¹H NMR (400 MHz, CDCl₃): δ 1.85 (s, 3H,NAc), 2.06 (s, 3H, OAc), 2.08 (s, 3H, OAc), 2.09 (s, 3H, OAc), 3.84 (s,3H, COOCH₃), 4.16-4.28 (m, 2H, H-5, H-9), 4.68 (dd, J=12.5, 2.6 Hz, 1H,H-9′), 4.91 (dd, J=10.7, 1.8 Hz, 1H, H-6), 5.43 (ddd, J=6.7, 5.6, 2.6Hz, 1H, H-8), 5.53 (dd, J=5.8, 1.7 Hz, 1H, H-7), 6.00 (dd, J=9.9, 2.5Hz, 1H, H-4), 6.11 (d, J=2.2 Hz, 1H, H-3), 6.59 (d, J=8.4 Hz, 1H, NH),7.20 (ddd, J=8.1, 7.3, 1.6 Hz, 1H, PH—H), 7.40 (td, J=7.5, 1.3 Hz, 1H,Ph-H), 7.63 (dd, J=8.0, 1.2 Hz, 1H, Ph-H), 7.96 (dd, J=7.9, 1.7 Hz, 1H,Ph-H), 8.22 (s, 1H, triazole-CH); ¹³C NMR (101 MHz, CDCl₃) (20.80, 20.95(3 OCOCH₃ ), 23.13 (NHCOCH₃ ), 49.56 (C-5), 52.75 (COOCH₃ ), 57.49(C-4), 62.10 (C-9), 67.81 (C-7), 70.62 (C-8), 76.10 (C-6), 107.03 (C-3),121.42 (Ph q carbon), 122.66 (Ph), 127.76 (Ph), 129.75 (Ph), 130.65(Ph), 133.59 (triazole-C5), 145.61 (triazole-C4), 145.94 (C-2), 161.33(COOCH₃), 170.15, 170.29, 170.71, 171.09 (NHCOCH₃, 3 OCOCH₃).

Sodium5-acetamido-2,6-anhydro-4-(4-(2-bromophenyl)-[1,2,3,]triazol-1-yl)-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(IE1257-84)

Yield=89%. ¹H NMR (400 MHz, D₂O): δ 1.93 (s, 3H, NAc), 3.62-3.77 (m, 2H,H-7, H-9), 3.92 (dd, J=12.0, 2.7 Hz, 1H, H-9′), 4.02 (ddd, J=9.2, 6.2,2.7 Hz, 1H, H-8), 4.49 (dd, J=10.9, 9.6 Hz, 1H, H-5), 4.60 (dd, J=10.9,1.3 Hz, 1H, H-6), 5.58 (dd, J=9.7, 2.3 Hz, 1H, H-4), 5.92 (d, J=2.3 Hz,1H, H-3), 7.36 (ddd, J=7.9, 7.3, 1.7 Hz, 1H, Ph-H), 7.49 (td, J=7.6, 1.3Hz, 1H, Ph-H), 7.69 (dd, J=7.8, 1.7 Hz, 1H, Ph-H), 7.77 (dd, J=8.1, 1.2Hz, 1H, Ph-H), 8.45 (s, 1H, triazole-CH); ¹³C NMR (101 MHz, D₂O): δ21.77 (NHCOCH₃ ), 48.68 (C-5), 60.07 (C-4), 63.10 (C-9), 68.11 (C-7),69.75 (C-8), 75.42 (C-6), 101.91 (C-3), 121.49 (Ph q carbon), 123.72(Ph), 127.90 (Ph), 129.16 (Ph), 130.16 (Ph q carbon), 130.42 (Ph),130.71 (Ph), 133.42 (triazole-C5), 145.89 (triazole-C4), 150.39 (C-2),168.78 (COONa), 173.54 (NHCOCH₃).

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(4-(2-methoxycarbonyl)phenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1398-25)

Purification by silica gel chromatography using hexane:acetone (4:3)yielded (91%) of pure IE1398-25. ¹H NMR (400 MHz, CDCl₃): δ 1.76 (s, 3H,NAc), 2.02 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.05 (s, 3H, OAc), 3.75 (s,3H, Ph-COOCH₃), 3.80 (s, 3H, C2-COOCH₃), 4.17 (dd, J=12.4, 7.1 Hz, 1H,H-9), 4.31 (q, J=9.8 Hz, 1H, H-5), 4.69 (dd, J=12.5, 2.8 Hz, 1H. H-9′),4.78 (d, J=10.3 Hz, 1H, H-6), 5.38 (ddd, J=7.6, 5.4, 2.8 Hz, 1H, H-8),5.52 (dd, J=5.0, 1.9 Hz, 1H, H-7), 5.78 (dd, J=10.2, 2.5 Hz, 1H, H-4),6.03 (d, J=2.4 Hz, 1H, H-3), 7.13 (d, J=8.9 Hz, 1H, NH), 7.38 (t, J=7.8Hz, 1H, Ph-H), 7.50 (td, J=7.7, 1.4 Hz, 1H, Ph-H), 7.65 (d, J=7.7 Hz,1H, Ph-H), 7.76 (d, J=7.6 Hz, 1H, Ph-H), 7.84 (s, 1H, triazole-CH); ¹³CNMR (101 MHz, CDCl₃) δ 20.72, 20.79, 20.91 (3 OCOCH₃ ), 22.80 (NHCOCH₃), 48.65 (C-5), 52.29, 52.66 (2 COOCH₃ ), 57.96 (C-4), 62.23 (C-9),67.78 (C-7), 70.91 (C-8), 76.66 (C-6), 107.37 (C-3), 122.06 (Ph), 128.35(Ph), 129.85 (Ph), 129.99 (Ph q carbon), 130.15 (Ph q carbon), 130.35(Ph), 131.62 (triazole-C5), 145.79 (triazole-C4), 145.89 (C-2), 161.36(C2-COOCH₃), 168.39 (Ph-COOCH₃), 170.07, 170.27, 170.76, 171.02(NHCOCH₃, 3 OCOCH₃); LRMS [C₂₈H₃₂N₄O₁₂] (m/z): (+ve ion mode) 639.3[M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(4-(2-methoxycarbonyl)phenyl)-[1,2,3,]triazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE1398-33)

Yield=68%. ¹H NMR (400 MHz, D₂O): δ 1.94 (s, 3H, NAc), 3.66 (dd, J=12.2,6.0 Hz, 1H, H-9), 3.72 (dd, J=9.8, 1.4 Hz, 1H, H-7), 3.93 (dd, J=12.0,2.9 Hz, 1H, H-9′), 4.03 (ddd, J=9.4, 6.4, 2.7 Hz, 1H, H-8), 4.47 (t,J=10.3 Hz, 1H, H-5), 4.61 (dd, J=10.9, 1.4 Hz, 1H, H-6), 5.59 (dd,J=9.5, 2.1 Hz, 1H, H-4), 5.95 (d, J=2.2 Hz, 1H, H-3), 7.43-7.55 (m, 3H,3 Ph-H), 7.75 (dd, J=6.6, 1.5 Hz, 1H, Ph-H), 8.18 (s, 1H, triazole-CH);¹³C NMR (101 MHz, D₂O): δ 21.76 (NHCOCH₃ ), 48.80 (C-5), 59.69 (C-4),63.10 (C-9), 68.13 (C-7), 69.77 (C-8), 75.40 (C-6), 102.22 (C-3), 122.33(triazole-C5), 125.00 (Ph q carbon), 126.44 (Ph), 128.30 (Ph), 128.57(Ph), 128.79 (Ph), 138.95 (Ph q carbon), 146.15 (triazole-C4), 150.22(C-2), 168.85 (C2-COONa), 173.80 (NHCOCH₃), 178.10 (Ph-COONa); LRMS[C₂₀H₂₀N₄Na₂O₉] (m/z): (+ve ion mode) 528.8 [M+Na]⁺.

Methyl7,8,9-tri-O-acetyl-3,4,5-trideoxy-3-fluoro-5-isobutyramido-4-(4-methoxymethyl-[1,2,3,]triazol-1-yl)-D-erythro-3-L-gluco-non-2-ulopyranosylonatefluoride (IE1257-22)

Purification by silica gel chromatography using hexane/acetone (3:2)yielded (57 mg, 81%) of pure 10. ¹H NMR (400 MHz, CDCl₃): δ 0.96-1.01(m, 6H, isobut-2CH₃), 2.01 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.13 (s, 3H,OAc), 2.23 (dq, J=13.0, 6.8 Hz, 1H, isobut-CH), 3.36 (s, 3H, OCH₃),3.90-3.96 (m, 4H, COOCH₃, H-5), 4.19 (dd, J=12.6, 4.6 Hz, 1H, H-9), 4.27(dd, J=12.6, 2.5 Hz, 1H, H-9′), 4.54 (s, 2H, OCH₂), 4.99-5.30 (m, 3H,H-3, H-6, H-7), 5.37 (ddd, J=8.6, 4.6, 2.4 Hz, 1H, H-8), 6.19-6.29 (m,2H, H-4, NH), 7.60 (s, 1H, triazole-CH); ¹³C NMR (100 MHz, CDCl₃): δ18.86 (isobut-CH₃), 19.32 (isobut-CH₃), 20.59, 20.75, 20.79 (3 OCOCH₃ ),35.53 (isobut-CH), 50.84 (d, J=5.8 Hz, C-5), 53.62 (COOCH₃ ), 58.34(OCH₃), 59.00-59.40 (m, C-4), 61.86 (C-9), 65.50 (OCH₂), 67.08 (C-7),68.29 (C-8), 71.51 (C-6), 90.64 (dd, J=196.1, 30.3 Hz, C-3), 105.58 (dd,J=230.3, 26.8 Hz, C-2), 124.52 (triazole-CH), 144.83 (triazole-qcarbon), 164.51 (d, J=32.9 Hz, COOCH₃), 169.56, 170.41, 170.69 (3OCOCH₃), 178.35 (isobut-CO); ¹⁹F NMR (376 MHz, CDCl₃): δ −118.67 (d,J=14.0 Hz, F−2α), −197.42 (d, J=13.2 Hz, F-3β); LRMS [C₂₄H₃₄F₂N₄O₁₁](m/z): (+ve ion mode) 615.3 [M+Na]⁺.

3,4,5-Trideoxy-3-fluoro-5-isobutyramido-4-(4-methoxymethyl-[1,2,3,]triazol-1-yl)-D-erythro-β-L-gluco-non-2-ulopyranosonicfluoride (IE1257-24)

¹H NMR (400 MHz, D₂O): δ 0.81 (d, J=6.9 Hz, 3H, isobut-CH₃), 0.94 (d,J=6.9 Hz, 3H, isobut-CH₃), 2.35 (p, J=6.9 Hz, 1H, isobut-CH), 3.38 (s,3H, OCH₃), 3.54 (d, J=9.2 Hz, 1H, H-7), 3.61 (m, 1H, H-9), 3.78-3.91 (m,2H, H-8, H-9′), 4.52-4.76 (m, 3H, OCH₂, H-6), 4.85 (m, 1H, H-5), 5.32(ddd, J=49.5, 13.7, 9.7 Hz, 1H, H-3), 5.75 (q, J=11.2 Hz, 1H, H-4), 8.28(s, 1H, triazole-CH); ¹³C NMR (100 MHz, D₂O): δ 18.11 (isobut-CH₃),18.65 (isobut-CH₃), 34.87 (isobut-CH), 48.15 (d, J=6.1 Hz, C-5), 57.26(OCH₃), 63.16 (C-9), 63.18-63.58 (m, C-4), 64.13 (OCH₂), 67.85 (C-7),69.83 (C-8), 73.73 (d, J=3.3 Hz, C-6), 90.22 (dd, J=190.9, 32.6 Hz,C-3), 106.79 (dd, J=224.1, 27.8 Hz, C-2), 124.89 (triazole-CH), 144.09(trizaole-q C), 169.21 (d, J=30.7 Hz, COOH), 180.88 (isobut-CO); ¹⁹F NMR(376 MHz, D₂O): δ −112.75 (d, J=13.8 Hz, F-2α), −199.41 (d, J=14.3 Hz,F-3β); LRMS [C₁₇H₂₅F₂N₄NaO₈] (m/z): (+ve ion mode) 496.8 [M+Na]⁺; HRMS(API) (m/z): [M+1]⁺ calcd for C₁₇H₂₆F₂N₄O₈[M+1]⁺453.1791. found,453.1810.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-(1-cyano-2H-isoindol-2-yl)-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(IE889-76)

¹H NMR (300 MHz, CDCl₃): δ 1.60 (s, 3H, NAc), 1.82 (s, 6H, 2 OAc), 1.84(s, 3H, OAc), 3.60 (s, 3H, COOCH₃), 3.96 (dd, J=12.7, 7.0 Hz, 1H, H-9),4.19 (q, J=9.9 Hz, 1H, H-5), 4.40-4.50 (m, 2H, H-6, H-9′), 5.16 (ddd,J=7.5, 6.0, 2.4 Hz, 1H, H-8); 5.22-5.33 (m, 2H, H-4, H-7), 5.86 (d,J=1.6 Hz, 1H, H-3), 6.20 (d, J=9.6 Hz, 1H, NHAc), 6.84 (m, 1H, Ar—H),6.99 (m, 1H, Ar—H), 7.26 (s, 1H, Ar—H-3′), 7.37 (m, 2H, Ar—H-4′,Ar—H-7′); ¹³H NMR (75 MHz, CDCl₃): δ 20.76, 20.92 (3 OCOCH₃ ), 22.97(NHCOCH₃ ), 49.26 (C-5), 52.79 (COOCH₃), 58.00 (C-4), 62.08 (C-9), 67.67(C-7), 70.86 (C-8), 77.06 (C-6), 93.82 (Ar—C—CN), 107.66 (C-3), 114.69(CN), 117.55 (Ar), 117.78 (Ar), 121.14 (Ar), 123.23 (Ar), 124.63 (Ar qcarbon), 126.08 (Ar), 131.60 (Ar q carbon), 146.63 (C-2), 161.29(COOCH₃), 170.17, 170.30, 170.63 (NHCOCH₃, 3 OCOCH₃); LRMS [C₂₇H₂₉N₃O₁₀](m/z): (+ve ion mode) 578.1 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-4-(1-cyano-2H-isoindol-2-yl)-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(IE889-80)

¹H NMR (300 MHz, D₂O): δ 1.91 (s, 3H, NAc), 3.61-3.77 (m, 2H, H-7, H-9),3.94 (dd, J=11.9, 2.6 Hz, 1H, H-9′), 4.05 (ddd, J=9.2, 6.2, 2.6 Hz, 1H,H-8), 4.50 (m, 1H, H-5), 4.64 (d, J=11.0 Hz, 1H, H-6), 5.47 (dd, J=9.4,2.3 Hz, 1H, H-4), 5.96 (d, J=2.3 Hz, 1H, H-3), 7.21 (m, 1H, Ar—H), 7.37(ddd, J=8.9, 6.7, 1.1 Hz, 1H, Ar—H), 7.65-7.87 (m, 3H, Ar—H-3′, Ar—H-4′,Ar—H-7′); ¹³C NMR (75 MHz, D₂O): δ 21.77 (NHCOCH₃ ), 49.83 (C-5), 59.20(C-4), 63.08 (C-9), 68.09 (C-7), 69.77 (C-8), 75.41 (C-6), 92.08(Ar—C—CN), 103.30 (C-3), 115.30 (CN), 117.35 (Ar), 119.65 (Ar), 121.36(Ar), 122.82 (Ar), 124.09 (Ar q carbon), 125.99 (Ar), 132.03 (Ar qcarbon), 150.56 (C-2), 168.78 (COONa), 173.36 (NHCOCH₃); LRMS[C₂₀H₂₀N₃NaO₇] (m/z): (+ve ion mode) 460.1 [M+Na]⁺; HRMS (API) (m/z):[M+Na]⁺ calcd for C₂₀H₂₀N₃Na₂O₇[M+Na]⁺460.1091. found, 460.1097.

Methyl7,8,9-tri-O-acetyl-2,6-anhydro-4-(1-cyano-2H-isoindol-2-yl)-3,4,5-trideoxy-5-isobutyramido-D-glycero-D-galacto-non-2-enonate(IE889-92)

¹H NMR (300 MHz, CDCl₃): δ 0.92 (d, J=6.8 Hz, 3H, isobutyryl-CH₃), 1.00(d, J=6.8 Hz, 3H, isobutyryl-CH₃), 2.07 (s, 3H, OAc), 2.09 (s, 3H, OAc),2.10 (s, 3H, OAc), 2.27 (m, 1H, isobutyryl-CH), 3.86 (s, 3H, COOCH₃),4.23 (dd, J=12.4, 6.6 Hz, 1H, H-9), 4.50 (m, 1H, H-5), 4.68 (dd, J=12.5,2.7 Hz, 1H, H-9′), 4.77 (d, J=10.6 Hz, 1H, H-6), 5.40 (m, 1H, H-8), 5.51(dd, J=5.6, 1.6 Hz, 1H, H-7), 5.62 (dd, J=9.7, 2.4 Hz, 1H, H-4), 6.15(d, J=2.4 Hz, 1H, H-3), 6.24 (d, J=9.4 Hz, 1H, NH), 7.11 (dd, J=8.6, 6.7Hz, 1H, Ar—H), 7.28 (dd, J=8.4, 6.6 Hz, 1H, Ar—H), 7.50 (s, 1H,Ar—H-3′), 7.60-7.64 (m, 2H, Ar—H-4′, Ar—H-7′); ¹³H NMR (75 MHz, CDCl₃):δ 18.77, 19.05 (isobutyryl-2CH₃), 20.69, 20.73, 20.90 (3 OCOCH₃ ), 35.51(isobutyryl-CH), 49.11 (C-5), 52.78 (COOCH₃ ), 57.88 (C-4), 62.05 (C-9),67.61 (C-7), 70.80 (C-8), 77.01 (C-6), 93.83 (Ar—C—CN), 107.56 (C-3),114.80 (CN), 117.58 (Ar), 117.81 (Ar), 121.20 (Ar), 123.22 (Ar), 124.66(Ar q carbon), 126.15 (Ar), 131.56 (Ar q carbon), 146.50 (C-2), 161.31(COOCH₃), 170.10, 170.14, 170.57 (3 OCOCH₃), 177.07 (isobutyryl-CO);LRMS [C₂₉H₃₃N₃O₁₀] (m/z): (+ve ion mode) 606.4 [M+Na]⁺.

Sodium2,6-anhydro-4-(1-cyano-2H-isoindol-2-yl)-3,4,5-trideoxy-5-isobutyramido-D-glycero-D-galacto-non-2-enonate(IE889-99)

¹H NMR (300 MHz, D₂O): δ 0.88 (d, J=6.9 Hz, 3H, isobutyryl-CH₃), 0.97(d, J=6.9 Hz, 3H, isobutyryl-CH₃), 2.44 (m, 1H, isobutyryl-CH),3.62-3.75 (m, 2H, H-7, H-9), 3.93 (dd, J=12.0, 2.7 Hz, 1H, H-9′), 4.04(ddd, J=9.3, 6.3, 2.6 Hz, 1H, H-8), 4.56-4.66 (m, 2H, H-5, H-6), 5.47(dd, J=9.6, 2.4 Hz, 1H, H-4), 5.95 (d, J=2.2 Hz, 1H, H-3), 7.20 (ddd,J=7.8, 6.8, 1.0 Hz, 1H, Ar—H), 7.36 (ddd, J=8.3, 6.8, 1.0 Hz, 1H, Ar—H),7.70 (dd, J=8.6, 1.1 Hz, 1H, Ar—H), 7.74-7.87 (m, 2H, 2 Ar—H); ¹³C NMR(75 MHz, D₂O): δ 18.16, 18.59 (isobutyryl-2CH₃), 35.06 (isobutyryl-CH),49.13 (C-5), 59.30 (C-4), 63.07 (C-9), 68.20 (C-7), 69.81 (C-8), 75.45(C-6), 92.08 (Ar—C—CN), 103.34 (C-3), 115.47 (CN), 117.33 (Ar), 120.04(Ar), 121.38 (Ar), 122.81 (Ar), 124.04 (Ar q carbon), 126.01 (Ar),132.04 (Ar q carbon), 150.37 (C-2), 168.87 (COONa), 180.37(isobutyryl-CO); LRMS [C₂₂H₂₄N₃NaO₇] (m/z): (+ve ion mode) 488.1[M+Na]⁺; HRMS (API) (m/z): [M+Na]⁺ calcd forC₂₂H₂₄N₃Na₂O₇[M+Na]⁺488.1404. found, 488.1400.

Methyl7,8,9-tri-O-acetyl-2,6-anhydro-4-(1-cyano-2H-isoindol-2-yl)-3,4,5-trideoxy-5-(2,2,2-trifluoroacetamido)-D-glycero-D-galacto-non-2-enonate(IE927-93)

¹H NMR (300 MHz, CDCl₃): δ 2.07 (s, 3H, OAc), 2.08 (s, 3H, OAc), 2.10(s, 3H, OAc), 3.86 (s, 3H, COOCH₃), 4.22 (dd, J=12.5, 6.6 Hz, 1H, H-9),4.54 (q, J=9.9 Hz, 1H, H-5), 4.68 (dd, J=12.5, 2.6 Hz, 1H, H-9′), 4.82(dd, J=10.6, 1.9 Hz, 1H, H-6), 5.43 (td, J=6.0, 5.5, 2.6 Hz, 1H, H-8),5.52 (dd, J=5.6, 1.8 Hz, 1H, H-7), 5.62 (dd, J=9.7, 2.4 Hz, 1H, H-4),6.17 (d, J=2.4 Hz, 1H, H-3), 7.13 (ddd, J=7.9, 6.8, 1.0 Hz, 1H, Ar—H),7.28 (ddd, J=8.4, 6.7, 1.0 Hz, 1H, Ar—H), 7.52 (s, 1H, Ar—H-3′),7.58-7.69 (m, 2H, Ar—H-4′, Ar—H-7′), 7.91 (d, J=9.6 Hz, 1H, NH); ¹³H NMR(75 MHz, CDCl₃): δ 20.50, 20.71, 20.91 (3 OCOCH₃ ), 49.81 (C-5), 52.93(COOCH₃ ), 57.45 (C-4), 61.93 (C-9), 67.46 (C-7), 70.64 (C-8), 76.40(C-6), 93.61 (Ar—C—CN), 107.14 (C-3), 113.24-117.05 (CF₃), 114.37 (CN),117.73 (Ar), 117.80 (Ar), 121.08 (Ar), 123.65 (Ar), 124.93 (Ar qcarbon), 126.55 (Ar), 131.85 (Ar q carbon), 146.73 (C-2), 157.56 (q,J_(C,F)=38.8 Hz, COCF₃), 161.03 (COOCH₃), 169.91, 170.38, 170.71 (3OCOCH₃); LRMS [C₂₇H₂₆—F₃N₃O₁₀] (m/z): (+ve ion mode) 632.1 [M+Na]⁺.

Sodium2,6-anhydro-4-(1-cyano-2H-isoindol-2-yl)-3,4,5-trideoxy-5-(2,2,2-trifluoroacetamido)-D-glycero-D-galacto-non-2-enonate(IE927-99)

¹H NMR (300 MHz, D₂O): δ δ 3.60-3.75 (m, 2H, H-7, H-9), 3.93 (dd,J=11.9, 2.7 Hz, 1H, H-9′), 4.07 (ddd, J=9.4, 6.4, 2.6 Hz, 1H, H-8),4.64-4.74 (m, 2H, H-5, H-6), 5.57 (dd, J=9.4, 2.3 Hz, 1H, H-4), 6.00 (d,J=2.3 Hz, 1H, H-3), 7.21 (m, 1H, Ar—H), 7.36 (m, 1H, Ar—H), 7.69 (d,J=8.6 Hz, 1H, Ar—H), 7.77 (d, J=8.5 Hz, 1H, Ar—H), 7.84 (s, 1H,Ar—H-3′); ¹³C NMR (75 MHz, D₂O): 50.48 (C-5), 58.73 (C-4), 63.00 (C-9),68.08 (C-7), 69.66 (C-8), 75.05 (C-6), 91.94 (Ar—Ĉ—CN), 103.21 (C-3),109.60-118.88 (CF₃), 115.01 (CN), 117.34 (Ar), 121.36 (Ar), 123.02 (Ar),124.19 (Ar q carbon), 126.23 (Ar), 132.12 (Ar q carbon), 150.62 (C-2),158.20 (q, J_(C,F)=38.8 Hz, COCF₃), 168.44 (COONa); LRMS[C₂₀H₁₇F₃N₃NaO₇] (m/z): (+ve ion mode) 514.1 [M+Na]⁺, 492.2; HRMS (API)(m/z): [M+Na]⁺ calcd for C₂₀H₁₇F₃N₃Na₂O₇[M+Na]⁺514.0830. found,514.0836.

Methyl5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-4-(1H-tetrazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-86)

¹H NMR (300 MHz, CDCl₃): δ 1.95 (s, 3H, NAc), 2.06 (s, 3H, OAc), 2.09(s, 3H, OAc), 2.10 (s, 3H, OAc), 3.84 (s, 3H, COOCH₃), 4.02 (m, 1H,H-5), 4.22 (dd, J=12.6, 6.4 Hz, 1H, H-9), 4.63 (dd, J=12.5, 2.6 Hz, 1H,H-9′), 4.95 (dd, J=10.5, 1.8 Hz, 1H, H-6), 5.38 (td, J=6.2, 2.6 Hz, 1H,H-8), 5.49 (dd, J=5.9, 1.8 Hz, 1H, H-7), 6.05 (d, J=2.5 Hz, 1H, H-3),6.11 (dd, J=9.8, 2.5 Hz, 1H, H-4), 6.67 (d, J=8.2 Hz, 1H, NH), 8.70 (s,1H, tetrazole-CH); ¹³H NMR (75 MHz, CDCl₃): δ 20.72, 20.75, 20.91 (3OCOCH₃ ), 23.13 (NHCOCH₃ ), 49.72 (C-5), 52.87 (COOCH₃ ), 55.92 (C-4),61.93 (C-9), 67.62 (C-7), 70.50 (C-8), 75.70 (C-6), 105.19 (C-3), 140.84(Tetrazole-C5), 146.64 (C-2), 161.07 (COOCH₃), 170.17, 170.27, 170.63,171.54 (NHCOCH₃, 3 OCOCH₃); LRMS [C₁₉H₂₅N₅O₁₀] (m/z): (+ve ion mode)506.5 [M+Na]⁺.

Sodium5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-(1H-tetrazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE832-98)

¹H NMR (300 MHz, D₂O): δ 1.94 (s, 3H, NAc), 3.65-3.74 (m, 2H, H-7, H-9),3.92 (dd, J=11.9, 2.7 Hz, 1H, H-9′), 4.02 (ddd, J=9.3, 6.2, 2.7 Hz, 1H,H-8), 4.44 (dd, J=10.9, 9.7 Hz, 1H, H-5), 4.62 (dd, J=11.0, 1.4 Hz, 1H,H-6), 5.73 (dd, J=9.6, 2.3 Hz, 1H, H-4), 5.88 (d, J=2.3 Hz, 1H, H-3);¹³C NMR (75 MHz, D₂O): δ 21.63 (NHCOCH₃ ), 48.44 (C-5), 58.77 (C-4),63.04 (C-9), 67.99 (C-7), 69.67 (C-8), 75.29 (C-6), 100.61 (C-3), 143.99(Tetrazole-C5), 150.85 (C-2), 168.53 (COOCH₃), 173.82 (NHCOCH₃); LRMS[C₁₂H₁₆N₅NaO₇] (m/z): (+ve ion mode) 388.4 [M+Na]⁺; HRMS (API) (m/z):[M+Na]⁺ calcd for C₁₂H₁₆N₅Na₂O₇[M+Na]⁺ 388.083964. found, 388.084945.

Methyl7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(1H-tetrazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE889-36)

¹H NMR (300 MHz, CDCl₃): δ 1.05 (d, J=6.9 Hz, 6H, isobutyramido-2CH₃),2.01 (s, 3H, OAc), 2.05 (s, 6H, 20Ac), 2.34 (m, 1H, isobutyramido-CH),3.81 (s, 3H, COOCH₃), 4.01 (m, 1H, H-5), 4.18 (dd, J=12.5, 6.3 Hz, 1H,H-9), 4.60 (dd, J=12.6, 2.6 Hz, 1H, H-9′), 5.03 (dd, J=10.5, 1.7 Hz, 1H,H-6), 5.32 (td, J=6.1, 2.5 Hz, 1H, H-8), 5.46 (dd, J=5.9, 1.8 Hz, 1H,H-7), 6.03 (d, J=2.4 Hz, 1H, H-3), 6.14 (dd, J=9.8, 2.5 Hz, 1H, H-4),6.94 (d, J=8.1 Hz, 1H, NH), 8.65 (s, 1H, tetrazole-CH); ¹³H NMR (75 MHz,CDCl₃): δ 18.85, 19.27 (isobutyramido-2CH₃), 20.66, 20.84 (3 OCOCH₃),35.56 (isobutyramido-CH), 49.69 (C-5), 52.79 (COOCH₃ ), 55.77 (C-4),61.91 (C-9), 67.49 (C-7), 70.51 (C-8), 75.64 (C-6), 105.15 (C-3), 140.81(Tetrazole-C5), 146.70 (C-2), 161.18 (COOCH₃), 170.04, 170.15, 170.58 (3OCOCH₃), 178.53 (isobutyramido-CO); LRMS [C₂₁H₂₉N₅O₁₀] (m/z): (+ve ionmode) 534.3 [M+Na]⁺; HRMS (API) (m/z): [M+Na]⁺ calcd for C₂₁H₂₉N₅NaO₁[M+Na]⁺534.180663. found, 543.1788531.

Sodium2,6-anhydro-3,4,5-trideoxy-5-isobutyramido-4-(H-tetrazol-1-yl)-D-glycero-D-galacto-non-2-enonate(IE889-42)

¹H NMR (300 MHz, D₂O): δ 0.99-1.04 (m, 6H, isobutyramido-2CH₃), 2.46 (m,1H, isobutyramido-CH), 3.59-3.75 (m, 2H, H-7, H-9), 3.93 (dd, J=12.0,2.6 Hz, 1H, H-9′), 4.03 (ddd, J=9.7, 6.2, 2.6 Hz, 1H, H-8), 4.49 (dd,J=10.9, 9.7 Hz, 1H, H-5), 4.65 (dd, J=10.9, 1.2 Hz, 1H, H-6), 5.76 (dd,J=9.7, 2.3 Hz, 1H, H-4), 5.88 (d, J=2.2 Hz, 1H, H-3), 9.38 (s, 1H,tetrazole-CH); LRMS [C₁₄H₂₀N₅NaO₇] (m/z): (+ve ion mode) 416.0 [M+Na]⁺;HRMS (API) (m/z): [M+Na]⁺ calcd for C₁₄H20N₅Na₂O₇ [M+Na]⁺416.115264.found, 416.116886.

Biology

Cells and Virus:

A549 cells (adenocarcinomic human alveolar basal epithelial cells) wereprovided by the European Collection of Cell Cultures (86012804-1VL,Sigma Aldrich). Cells were propagated in Dulbecco's Modified EagleMedium (DMEM) (Lonza, Basel, Switzerland) supplemented with 1% Glutamine(200 mM) and 5% foetal bovine serum. For infection and post-infectionprocedures, A549 cells were maintained in DMEM supplemented with 1%Glutamine only. Normal human bronchial/tracheal epithelial (NHBE) cells(CC-2540, lot 313831, Lonza) were amplified in B-ALI™ growth medium(Lonza) and the same medium was used for infection and post-infectionstudies. LLC-MK2 cells (Rhesus monkey kidney, ATCC CCL-7) were culturedin Eagle's minimal essential medium (EMEM) (Lonza) supplemented with 1%Glutamine (200 mM) and 2% of heat-inactivated foetal bovine serum.During hPIV-3 infection and post-infection incubation, LLC-MK2 cellswere maintained in EMEM supplemented with 1% glutamine. All cell lineswere incubated at 37° C. in a humidified atmosphere of 5% C02.

hPIV-3 (strain C-243) was obtained from the American Type CultureCollection (ATCC, Manassas, Va.). The virus was propagated in LLC-MK2cells with EMEM supplemented with glutamine (EMEM_(inf)) at 35° C. in ahumidified atmosphere of 5% CO₂. Virus-containing culture supernatantwas collected 3 to 4 days post-infection, while monitoring cytopathiceffects, and clarified from cell debris by centrifugation (3,000 RCF for15 min). Virus was concentrated at least 10 times using 30 kDa AmiconUltra filter unit (Millipore, Billerica, Mass.) for use inHaemagglutination Inhibition (HI) assays. Neuraminidase Inhibition (NI)assays and STD-NMR experiments used virus that was PEG-precipitated andthen purified as described above.

Clarified hPIV-3 supernatant was mixed with PEG6000 (8% finalconcentration) and NaCl (0.4 M final concentration) then incubatedovernight at 4° C. under gentle agitation. PEG6000/hPIV-3 complex waspelleted by centrifugation at 3,000 RCF for 30 min at 4° C. Thesupernatant was discarded and a volume of GNTE buffer (200 mM glycine,200 mM NaCl, 20 mM Tris-HCl, 2 mM EDTA, pH 7.4) corresponding to atleast 1:40 of the initial virus suspension volume was used to resuspendthe pellet overnight at 4° C. The virus suspension was homogenized by upand down pipetting followed by a mechanical disruption of the remainingvirus aggregates using a douncer with “tight” pestle. The hPIV-3homogenate was loaded on top of a 30%-60% non-linear sucrose gradientprepared in GNTE buffer and centrifuged at 100,000 RCF for 2 h 30 min at4° C. without brake for deceleration. The virus was concentrated at the30%-60% sucrose interface and then collected and stored at −80° C. forNI assays or at 4° C. for STD-NMR experiments.

hPIV-3 HN Inhibitors:

Compounds 2, 3, 5-10 were each provided as a lyophilized powder and thensolubilized in sterile water to generate a 10 mM stock solution.Solutions were sonicated for 15 min to allow complete dissolution andthen filter-sterilized. The stock solution was stored in a glass vial at−20° C. and freshly diluted in appropriate buffer before use. For STDNMR experiments, stock solutions were prepared in D₂O at 100 mM.Solutions were processed and stored as described above.

Recombinant HN Expression and Purification:

The HN protein was expressed using the Bac-to-Bac® baculovirusexpression system (Invitrogen, Carlsbad, Calif.) based on asubstantially modified literature procedure. Thus, the nucleotidesequence for a honeybee melittin signal peptide (HBM) was addeddownstream to the sequence encoding for the HN ectodomain (amino acids125 to 572). This sequence (HBM+HN) was codon optimised for expressionin Spodoptera frugiperda cells (Sf9) and ordered directly through theDNA2.0 gene synthesis service (DNA2.0, Menlo Park, Calif.) as a genenamed HBM-HNhPIV-3_(opt). HBM-HNhPIV-3_(opt) was amplified by PCR andligated into a pFastBacCT-TOPO® vector that provides an additionalC-terminal 6-histidine tag (His-Tag) for purification and detectionpurposes.

The generation and amplification of recombinant baculovirus containingHBM-HNhPIV-3_(opt) were performed according to the manufacturer'sinstructions. Sf9 cells (Invitrogen), cultured in Insect-XPRESS proteinfree insect cell medium (Lonza), were infected with high MOI ofHBM-HNhPIV-3_(opt) baculovirus. Four days post-infection thesupernatant, containing recombinant HN, was collected to yield thehighest protein expression. The supernatant was clarified bycentrifugation (3,000 RCF for 15 min) to remove cell debris and thenpurified on a HisTrap excel 5 mL column (GE Healthcare life sciences,Buckinghamshire, England) following the manufacturer's protocol.Recombinant HN was eluted with 500 mM imidazole solution and collectedfractions were assessed by a neuraminidase activity (NA) assay (seebelow). The most active fractions were pooled and concentrated with a 10kDa Amicon Ultra filter unit (Millipore) to a final volume of 800 μL. Anadditional purification step was performed that employed fast proteinliquid chromatography (Amersham Biosciences) over a Superdex 75 gelfiltration column (GE Healthcare) at 4° C. and 1 mL fractions werecollected with a Frac-920. Protein-containing fractions, as determinedby monitoring fraction collection at 280 nm, were assessed in a NA assayas well as subjected to SDS-PAGE. Purified and concentrated recombinantHN protein was stored at 4° C.

Haemagglutination Inhibition Assay:

The HN inhibitors were assessed in duplicate in a U-bottom 96 well plateassay. Compounds were diluted in PBS as a 4× solution for eachconcentration tested (25 μL/well, 1× final). Each dilution was mixedwith 4 haemagglutination units (HAU) of hPIV-3 (25 L/well, 1 HAU final)and incubated for 20 min at room temperature. The plate was transferredon ice and an equivalent volume (50 μL) of ice-cold 0.75% guinea pig redblood cells (Gp-RBC) or 1% human red blood cells (h-RBC) was added toeach well. The plate was then incubated for 1 h 30 min at 4° C. beforereading the extent of haemagglutination. The HI IC₅₀ was considered asthe concentration of inhibitor that reduced the haemagglutinin activity(agglutination) by 50% compared to those of a non-treated virussuspension.

Neuraminidase Inhibition Assay

Purified hPIV-3, inhibitors and MUN were prepared and diluted in NAReaction Buffer (NaOAc 50 mM, CaCl₂ 5 mM, pH 4.6). NA, employingdifferent hPIV-3 dilutions, were initially measured to determine thelowest virus concentration to be used in the assays. The NA assays wereperformed with enough purified virus to obtain a maximal fluorescencesignal at least 5 times higher than the background for the experiment tobe considered statistically significant. Neuraminidase inhibition (NI)assays were done in triplicate. For each concentration tested, 2 μL ofpurified hPIV-3 and 4 μL of 2.5× inhibitor solution (1× final) was addedto each well. The plate was kept at room temperature for 20 min before 4μL of 5 mM 2′-(4-methylumbelliferyl) □-D-N-acetylneuraminide (MUN) (2 mMfinal) was added to each well and then the plate incubated at 37° C. for30 min with agitation (1000 rpm). The enzymatic reaction was stopped bythe addition of 190 μL of glycine buffer (glycine 0.25 M, pH 10.4) toeach well. A negative control was included by the addition of MUN tovirus and then the enzymatic reaction stopped at t=0. Relativefluorescence (RF) was measured with a Victor 3 multilabel reader(PerkinElmer, Waltham, Mass.). Data were processed by backgroundsubtraction (negative control RF) and then analysed with GraphPadPrism 4(GraphPad Software Inc., La Jolla, Calif.) to calculate IC₅₀ values(nonlinear regression (curve fit), Dose-response—inhibition, 3 parameterlogistic). The concentration of inhibitor that reduced neuraminidaseactivity (relative fluorescence) by 50% compared to those of anon-treated virus suspension was considered to be the NI IC₅₀ value.K_(i) values of inhibitors 6 and 10 were determined by enzyme kineticexperiments with whole hPIV-3 virus based on previously publishedprocedures. Thus, neuraminidase activity was measured every 5 min over a20 min period, at five substrate concentrations [S]: 2, 4, 8, 10 and 16mM, and four inhibitor concentrations [I]: 0, 0.5, 2.5 and 5 μM for 10or 0, 10, 20 and 60 μM for 6. All assays were performed in triplicateand the final data were fitted to the Michaelis-Menten equation forcompetitive inhibition using GraphPadPrism 4 (GraphPad Software Inc., LaJolla, Calif.) to determine the Michaelis-Menten constant (K_(m)), usingdata from the [I]=0 and variable [S] experiments, and K_(i) values.

Virus Growth Inhibition Assay:

Before assessing the best inhibitors in cell-based assays, an MTT assaywas performed to evaluate compound cytotoxicity. No cytotoxic effect wasobserved after incubation for 48 h of LLC-MK2 cells with 6, 8 and 10 at30 μM, the highest concentration tested. Virus growth inhibition wasassessed using a focus-forming assay by titration of progeny in thepresence of 2 μM of 6, 8 and 10 in EMEM_(inf) from a low MOI infectedconfluent LLC-MK2 monolayer in a 48 well plate format. Virus inoculum(100 FFU/well) was pre-incubated with 6, 8 and 10 for 20 min. Infectionwas performed in duplicate and continued for 1 h at 37° C. with gentleagitation every 15 min. Inocula were removed and replaced with 500μL/well of each respective 2 μM compound dilution (in EMEM_(inf)).

A positive control for infection was included using the same conditionsminus the compound. Virus proliferation on infected cell monolayers weremaintained for 48 h at 37° C., 5% CO₂. Culture supernatants fromduplicates were collected, pooled and clarified at 15,000 RCF for 10 minand stored at −80° C. Supernatants were diluted in EMEM_(inf) by 10⁻³,10⁻⁴ and 10⁻⁵ to avoid any remaining compound effect on the subsequentvirus titration. Virus titrations were done in duplicate using thepreviously described conditions for virus infection. After 1 h, Avicel(FMC BioPolymer, Philadelphia, Pa.) in EMEM_(inf) was directly added tothe inoculum (1% final concentration) to restrict and localise virusproliferation. The plate was incubated for 36 to 40 h at 37° C., 5% CO₂to allow focus formation. Avicel was gently removed and replaced with3.7% Paraformaldehyde/PBS and the plate was then kept for 15 min at roomtemperature for virus inactivation and cell fixation. Cell monolayerswere washed three times for 5 min each with PBS and then endogenousperoxidase inactivated with 0.3% H₂O₂/PBS for 30 min at 37° C. The platewas washed again three times for 5 min each with PBS and incubated withmouse monoclonal IgG anti-hPIV-3 HN (Fitzgerald, clone# M02122321, 2.0mg/mL) at 1 μg/mL in 5% milk/PBS for 1 h at 37° C. Cell monolayers werewashed 3 times for 5 min with 0.02% Tween20/PBS. Goatanti-Mouse-IgG(H+L)-HRP conjugate (BioRad, ref#170-6516) diluted at1:1000 in 5% milk/PBS was added to each well and incubated for 1 h at37° C. Cell monolayers were washed as previously described with 0.02%Tween20/PBS and then rinsed twice with PBS. Foci were revealed by addingTrueBlue solution (HRP substrate) on each well and incubating the platefor 1 h at 37° C. The TrueBlue solution was discarded and the platerinsed twice with water then dried before being scanned (FIG. 2) andfoci counted. The IC₅₀ value was considered as the concentration ofinhibitor that reduced the progeny virus titre by 50% compared to anon-treated infected LLC-MK2 monolayer.

In situ ELISA:

In situ ELISA is a useful technique to evaluate virus growth inhibition.It measures, in one step, the expression level of hPIV-3 HN at the cellsurface of an infected cell monolayer. The expression level is directlycorrelated to the ability of a non-immobilized virus to infect andre-infect target cells. Infection was performed on a confluent cellmonolayer seeded in a 96 well plate. Virus (40 FFU/well) waspre-incubated for 20 min with compound 6 and 10 at a final concentrationfrom 1000 μM to 0.001 μM as a 10-fold dilution series. Infection wasdone in triplicate and continued for 1 h at 37° C. with gentle agitationevery 15 min. Inocula were removed and replaced with 200 μL/well of eachrespective compound dilution. A positive control for infection wasincorporated by the use of identical experimental conditions, minusinhibitor. Infected cell monolayers were kept for 36-40 h at 37° C., 5%CO₂ for virus proliferation. Virus was inactivated and cells fixed bythe direct addition of 100 μL of 11.1% paraformaldehyde/PBS. The platewas maintained at room temperature for 15 min and then washed 3 timesfor 5 min with PBS and then endogenous peroxidases were inactivated bytreatment with 0.3% H₂O₂/PBS for 30 min at 37° C. The cell monolayerswere washed and incubated with mouse monoclonal IgG anti-hPIV-3HN(Fitzgerald, clone# M02122321, 2.0 mg/mL) at 1 μg/mL in 5% milk/PBS for1 h at 37° C. The wells were washed 3 times for 5 min with 0.02%Tween20/PBS. Goat anti-Mouse-IgG(H+L)-HRP conjugate (BioRad,ref#170-6516), diluted at 1:2000 in 5% milk/PBS, was added to each welland incubated for 1 h at 37° C. Cell monolayers were washed with 0.02%Tween20/PBS and then rinsed twice with PBS. BD OptEIATMB substrate (BDBiosciences, San Jose, Calif., 100 μL) was added to each well and theplate was then incubated at 37° C. The enzymatic reaction was stoppedafter 3-5 min by the addition of 50 μL of 0.6 M of H₂SO₄ per well. Rawdata were obtained by reading the absorbance (OD) of each well at 450 nmfor 0.1 sec with a Victor 3 multilabel reader (PerkinElmer, Waltham,Mass.). Final ODs were obtained by subtraction of the negative control(non-infected cells) OD from the initial OD reading and the dataanalysed with GraphPadPrism4 (GraphPad Software Inc., La Jolla, Calif.)to calculate IC₅₀ values (nonlinear regression (curve fit),Dose-response—inhibition, 4 parameter logistic). The IC₅₀ value wasconsidered as the concentration of inhibitor that reduced the absorbanceat 450 nm by 50%, compared to a non-treated infected cell monolayer.

Compounds of the present invention can be tested in a hPIV-3 inhibitionassay on well-differentiated human airway epithelal (HAE) cells using apublished model. In brief the testing procedure is as follows: Humanairway epithelial (HAE) cells are isolated, cultured and differentiatedas previously described (Müller et al., 2013). Briefly, human nasalairway epithelial cells are isolated, expanded and seeded oncollagen-coated permeable membrane supports. Once the cells areconfluent, the apical medium is removed and cells are maintained at theair-liquid interface for approximately 4 to 6 weeks to allow epithelialdifferentiation. Cultures containing ciliated cells are inoculated viathe luminal surface with 5000 focus forming units of hPIV-3 per well for1 hour. Test compounds of formula (I), (II), (III) and (IIIa) at variousconcentrations are added to the basolateral medium just after the cellshave been infected with the virus. Viral load reduction is assessed at1, 3 and 6 days post-infection by virus titration using focus formingassay or in situ ELISA in A549 or LLC-MK2 cells, as previously published(Guillon et al., 2014). These results may be compared with a prior artreference compound such as a BCX compound including BCX-2855 to give anindication of relative potency (Guillon, P., Dirr, L., EI-Deeb, I. M.,Winger, M., Bailly, B., Haselhorst, T., Dyason, J. C., and von Itzstein,M. (2014). Structure-guided discovery of potent and dual-acting humanparainfluenza virus haemagglutinin-neuraminidase inhibitors. Nat.Commun. 5; and Müller, L., Brighton, L. E., Carson, J. L., Fischer, W.A., and Jaspers, I. (2013). Culturing of Human Nasal Epithelial Cells atAir Liquid Interface. J. Vis. Exp.)

Results

A comparison of the potency of the synthesised C4 modified Neu5Ac2enderivatives against hPIV-3 HN was undertaken and, for convenience sake,the IC₅₀ values were divided into two groups based on the acylaminogroup present at C5. Group 1 inhibitors have a C5 acetamidofunctionality and Group 2 inhibitors have a C5 isobutyramidofunctionality (FIG. 3 and FIG. 4A-C). The benchmark andwell-characterised broad spectrum neuraminidase inhibitor Neu5Ac2en (2)showed the weakest inhibition with IC₅₀ values of 1565 M and 1438 μM forhPIV-3 HN NI and HI, respectively. The inhibition observed for 3, the C5acetamido analogue of BCX 2798 (6), was improved when compared to 2,although it was still in the high micromolar range with IC₅₀ values of138 M and 210 μM for hPIV-3 HN NI and HI, respectively. These IC₅₀values were similar to those observed for our novel inhibitor 7, a C4methoxymethyl functionalised triazole Neu5Ac2en derivative, withexperimentally determined hPIV-3 HN NI and HI IC₅₀ values of 154 μM and313 μM, respectively. A significant improvement in potency was observedupon replacement of the C4 triazole's methoxymethyl moiety (7) with abulkier phenyl group (8). IC₅₀ values of 6.5 μM and 4.6 μM weredetermined for hPIV-3 HN NI and HI for 8, respectively. The values aresummarized in table 2.

In the second group of inhibitors, that contain a C5 isobutyramidofunctionality, it was obvious that the affinity of each inhibitor wasimproved relative to its C5 acetamido analogue. The order of potency,not unexpectedly, was identical within the same group. Thus, the weakestinhibition was found for 5, the C5 isobutyramido analogue of Neu5Ac2en,with IC₅₀ values of 188 M and 358 M for NI and HI respectively and IC₅₀values of 21.5 μM and 16.1 μM for NI and HI, respectively weredetermined for the reference hPIV inhibitor BCX 2798 (6). Inhibitor 9,with the relatively small methoxymethyl substituent on the triazolering, had IC₅₀ values close to those determined for the C4 azidoanalogue 6 (IC₅₀=14.2 μM and 13.8 μM for NI and HI respectively).Similarly as observed in the C5 acetamido-containing Group 1 inhibitors,increasing the substituent size from the methoxymethyl group ininhibitor 9 to a bulkier phenyl moiety as in inhibitor 10, resulted in aremarkable improvement in potency with IC₅₀ values of 2.7 μM and 1.5 μMfor NI and HI, respectively. Interestingly, an improvement in HI IC₅₀values was observed when human red blood cells were used instead ofguinea pig red blood cells (FIG. 2). This improvement most likelyreflects sialic acid content and/or linkage presentation differencesbetween human and guinea pig red blood cells. For example, it is wellknown that human tissues and cells, including red blood cells, onlyexpress N-acetylneuraminic acid-containing glycoconjugate receptors,whereas other animals also express N-glycolylneuraminic-acid-basedreceptors. Nevertheless, irrespective of the specific red blood cellsused, our designer inhibitor 10 had significantly higher potency whencompared to the benchmark compound 6. A K_(m) value for MUN of 5.1 mMand K_(i) values of 1.3 μM and 16 μM for inhibitor 10 and 6,respectively.

TABLE 2 NI and HI IC₅₀ values. Mean IC₅₀ values for each of the testedcompounds with calculated standard deviation and standard error. 2 3 7 8Inhibitor NI HI NI HI NI HI NI HI Mean IC₅₀ 1565 1438 138.1 210 154.4312.5 6.512 4.583 Std. deviation 439.1 427 55.69 65.19 18.93 25 0.63050.7217 Std. error 219.6 213.5 21.05 29.15 8.467 12.5 0.364 0.4167 5 6 910 Inhibitor NI HI NI HI NI HI NI HI Mean IC₅₀ 187.7 358.3 21.46 16.1214.16 13.75 2.74 1.458 Std. deviation 39.52 52.04 4.753 4.891 2.0284.787 0.2319 0.3608 Std. error 19.76 30.05 1.797 2.187 1.171 2.3940.1339 0.2083Cell-Based Assays

Following initial enzymatic screening, the most potent inhibitor 10 andthe reference hPIV inhibitor (BCX 2798, 6) were then evaluated in agrowth inhibition assay to compare their capacity to inhibit hPIV-3virus infection and propagation in LLC-MK2 cells (FIGS. 5A and B).Compound 6 was chosen as a reference inhibitor as it is the mostdocumented hPIV-3 Neu5Ac2en-based inhibitor to date and has reasonablein vitro hPIV-3 antiviral potency. In an initial assay, at an inhibitorconcentration of 2 μM, the virus was propagated for 48 h in the presenceof 6 or 10 and virus titres were determined. At this inhibitorconcentration, a reduction of 14% and 94% in virus titre by 6 and 10respectively was calculated (FIG. 5B). Virus growth inhibition IC₅₀values were then determined for the two inhibitors in a well-establishedin situ ELISA technique using three different cell lines. The LLC-MK2(monkey kidney epithelial cells) cell line was chosen as it isextensively used in hPIV-3 cell-based infection studies, as well as thehPIV-3 susceptible human respiratory cell lines A549 (lungadenocarcinoma epithelial cells) and normal human bronchial epithelial(NHBE) primary cells to investigate virus growth inhibition in naturaltissue-related cells. The method itself has useful advantages over thevirus titration method, as it is a faster, one-step, non-subjectivetechnique that correlates non-immobilized virus growth to HN expressionlevels of a low multiplicity of infection (MOI) infected cell monolayer.Interestingly, slightly lower virus growth inhibition IC₅₀ values weredetermined for 10 and 6 with the laboratory established cell lineLLC-MK2 in relation to the human cell lines. Overall, the same trend isobserved for all three cell lines in that a significantly strongerantiviral effect of inhibitor 10 (IC₅₀=2.1-13.9 μM) is determinedcompared to inhibitor 6 (IC₅₀=54.6-130.6 μM) (FIG. 6).

Structural Biology

Sample Preparation and ¹H NMR Experiments:

All NMR experiments were performed on a 600 MHz NMR spectrometer(Bruker) equipped with a 5-mm TXI probe with triple axis gradients.Intact virus suspension or recombinant hPIV-3 HN were buffer exchangedagainst 50 mM deuterated sodium acetate, 5 mM CaCl₂ in D₂O at pD 4.6 byultrafiltration using an Amicon Filter Unit (Millipore) with a cut-offvalue of 30 kDa or 10 kDa, respectively. For each experiment 20 μMhPIV-3 HN protein and a protein:ligand molar ratio of 1:100 in a finalvolume of 200 μL was used.

¹H NMR spectra were acquired with 32 scans at 283 K, a 2 s relaxationdelay over a spectral width of 6000 Hz. Due to the safer and easierhandling of protein compared to virus and in order to provide exactlythe same protein concentration in each experiment, the initial STD NMRexperiment was carried out for compound 10 in complex with intact hPIV-3virus, while all subsequent experiments were carried out using therecombinant HN protein.

Saturation Transfer Difference (STD) NMR Experiments:

The protein was saturated on-resonance at −1.0 ppm and off-resonance at300 ppm with a cascade of 60 selective Gaussian-shaped pulses of 50 msduration, resulting in a total saturation time of 3 s and the relaxationdelay was set to 4 s. Each STD NMR experiment was acquired either with atotal of 1056 scans (recombinant hPIV-3 HN) or 1512 scans (intact virus)and a WATERGATE sequence was used to suppress the residual HDO signal. ASpin-lock filter with 5 kHz strength and duration of 10 ms was appliedto suppress protein background. Control STD NMR experiments wereperformed with an identical experimental setup and the same ligandconcentration but in the absence of protein. On- and off-resonancespectra were stored and processed separately, and the final STD NMRspectra were obtained by subtracting the on-from the off-resonancespectra. All STD effects were quantified using the equationA_(STD)=(I₀−I_(sat))/I₀=I_(STD)/I₀. Therefore signal intensities of theSTD NMR spectrum (I_(STD)) were compared to the corresponding signalintensities of a reference spectrum (I₀). The strongest STD signal inthe spectrum was assigned to a value of 100% and used as a reference tocalculate relative STD effects accordingly.

Saturation Transfer Difference (STD) NMR experiments of 8 in complexwith recombinantly-expressed hPIV-3 HN (FIG. 7) and the most potentinhibitor 10 in complex with either recombinantly-expressed hPIV-3 HN(FIG. 8) or intact hPIV-3 virus (FIG. 9) were undertaken to furthersupport the computational and biological studies that demonstratedspecific binding and inhibition.

STD NMR signal intensities for all protons associated with 8 or 10 wereclearly observed, to varying extents, when the inhibitor is in complexwith either intact virus or recombinant hPIV-3 HN and clearlydemonstrated that the ligand binds in both instances. The minor signalsvisible at 3.25, 3.5 and 4.0 ppm in the ¹H NMR spectrum of 10 acquiredin the presence of intact virus particles were a consequence ofimpurities from the virus purification process and belong to neither thevirus particles nor 10. As anticipated, none of these signals wereobserved in the STD NMR spectrum and clearly demonstrate that theimpurities do not bind to the virus (FIG. 9). These experiments clearlydemonstrate the specific binding of 10 to both intact hPIV-3 virus andhPIV-3 HN, further substantiating the inhibitor's biological relevanceand potential.

Importantly, an overlay of the aromatic phenyl protons signals observedat 7.1-7.6 ppm in the STD NMR spectra for both the intact virus andrecombinant HN protein also reveals that the binding epitope ofinhibitor 10 is similar, if not identical, when bound either to intacthPIV-3 virus or to recombinant hPIV-3 HN protein (FIG. 10).

Epitope Mapping of Inhibitor 10

A complete ligand binding epitope was determined by the analysis of STDNMR spectra (FIG. 8) of hPIV-3 HN protein in complex with 10. All STDNMR signals of 10 were normalized to the strongest STD NMR signalobserved, the inhibitor's H4′ proton at 7.18 ppm. Relative STD NMReffects for all protons of the inhibitor were then calculated (Table 2).The extent of the STD NMR signal intensity strongly depends on theproton's proximity to the protein surface and reveals how the designedinhibitor 10 engages the HN protein's binding site.

TABLE 3 Relative STD NMR effects^(a) of 8 and 10 in complex with hPIV-3HN. Inhibitor 8 Inhibitor 10 (%) (%) Triazole CH 63 75 ArH2′ ArH6′ 92 95ArH3′ ArH5′ 100 100 ArH4′ 100 100 H3 85 80 H4 59 59 H5 50 49 H6 49 47 H730 36 H8 30 35 H9 12 24 H9′ 18 21 Isoprop-CH — 54 Isoprop-2CH₃ — 42 NHAc41 — ^(a)STD effects calculated according to the formula A_(STD) = (I₀ −I_(sat))/I₀ − I_(STD)/I₀. All STD NMR effects are given relative to thestrongest STD NMR intensity of the C4 triazolo ArH4′.

Notably, very strong relative STD NMR effects were observed for thephenyl group protons H2′, H3′, H4′, H5′ and H6′ between 7.1 ppm and 7.6ppm revealing a close contact in that region of the molecule to theprotein surface. Moreover, a significant STD NMR effect was likewisedetected for the CH of the triazole moiety. In contrast, the C5isobutyramido moiety's protons of the inhibitor showed less effect(relative STD NMR signal intensities in the range of 42-54%).

The protons associated with the Neu5Ac2en core structure of 10 displayedvariable relative STD NMR effects. A significant H3 relative STD NMRsignal intensity (80%) suggests a strong interaction of this part of themolecule with hPIV-3 HN. Furthermore, relative STD NMR signalintensities for H4, H5 and H6 of 59%, 50% and 49%, respectively,demonstrate that the ring protons of the Neu5Ac2en core structure arealso involved in inhibitor engagement to the protein.

Finally, weaker relative STD NMR effects of 36%, 35%, 24% and 21%, wereobserved for the glycerol side chain protons H7, H8, H9 and H9′,respectively and suggest that the glycerol sidechain makes less of acontribution to the inhibitor binding event compared with the C4triazolo functionality and the inhibitor's core ring structure (FIG. 8).The inhibitor 8 epitope map (FIG. 7) was for all intents and purposesidentical to that of inhibitor 10, with the C4 triazolo moiety clearlyin close contact to the protein surface.

Difluoro Analogues

The compounds may include diflouronated compounds and testing has beenperformed on select members of this class (I-170, I-179 and I-104 shownbelow). The target of such compounds is the haemagglutinin-neuraminidaseof hPIV-3 and hPIV-1. A co-crystal structure of hPIV-3 HN in complexwith 1-170 has been obtained. All of the below compounds have beentested in (i) NI enzymatic assays against hPIV-3 (and 170 also againsthPIV-1); (ii) cell based assays with the human cell line A549 cells(adenocarcinomic human alveolar basal epithelial cells) have beenevaluated using hPIV-3; (iii) cell cytotoxicity tests of the compoundsagainst A549 cells; and (iv) NI enzymatic assays against the humanNeuraminidase 2 showed no activity. No cell cytotoxicity was observedfor any of the compounds at 150 μM using A549 cells as shown in FIG. 11.No activity was observed against human Neu2 as indicated in FIG. 12.

TABLE 4 Enzymatic and cell-based assays for select difluorinatedcompounds with I-57 and I-40 as the corresponding non-fluorinated ‘en’compounds for comparison. I-170

I-179

I-104

μM 6 I-70 I-57 I-79 I-40 I-104 NI hPIV-3  18  6 2.5 63 12  4 IC₅₀Cell-based 130 14 10  45 80 25 ELISA IC₅₀Influenza Virus Sialidase Activity Assay

In a standard 96-well plate format, by use of sialidase from Influenza Aand B, the synthesized compounds can be assayed for their capacity toinhibit influenza virus sialidase by a modification (Biochim. Biophys.Acta 1991, 10, 65-71) of the fluorometric method of Potier et al. (Anal.Bio-chem. 1979, 94, 287-296) using the fluorogenic substrate4-methylumbelliferyl N-acetyl-α-D-neuraminide (MUN). All inhibitionassays can be done in triplicate over six inhibitor concentrations andat with 0.1 mM MUN. Specifically, 7 μL of 50 mM sodium acetate-6 mMCaCl2 buffer (pH 5.5) is added to each well of a 96-well solid blackplate on ice, followed by 1 μL of inhibitor, 1 μL of sialidase, andfinally 1 μL of the substrate MUN. The plate is then briefly centrifugedup to 1000 rpm for approximately 10 s to combine all components, and themixture can be incubated at 37° C. with 900 rpm shaking for 20 min. Tostop the reaction, 250 μL of 0.25 M glycine, pH 10, may be added to eachwell, and the fluorescence read (1 s per well) at an excitation of 355nm and emission of 460 nm with a Victor 3 multilabel reader(PerkinElmer, Waltham, Mass.). Data can be processed by backgroundsubtraction (negative control RF) and then analysed with GraphPadPrism 4(GraphPad Software Inc., La Jolla, Calif.) to calculate IC₅₀ values(nonlinear regression (curve fit), Dose-response—inhibition, 3 parameterlogistic).

In Situ Cellular ELISA for Influenza A and B

To evaluate virus growth inhibition of Influenza A and Influenza B virusfor the synthesised compounds, MDCK cells will be infected withinfluenza A or Influenza B virus in an in situ cellular ELISA developedbased on the principles described by Berkowitz and Levin, 1985(Antimicrob. Agents Chemother, 28, 207-210) and adapted to IAV by Myc etal, 1999 (J. Virol. Methods 77, 165-177 (1999)). MDCK cells in 100 μlEagle's Minimum Essential Medium (EMEM) supplemented with 2 mM glutamineand 10% FBS are seeded on flat-bottom 96-well microtiter plates andincubated overnight. On the next day, the culture medium is removed andcells washed with medium. A total of 50 μl of viral inoculum (40FFU/well) are added to the wells and incubated at 37° C., 5% CO₂ for 1 hwith gentle agitation every 15 minutes. The viral inoculum is thenremoved and replaced with 100 μl of infection medium (EMEM supplementedwith 2 mM glutamine and 3.0 μg/ml of TPCK treated trypsin). InfectedMDCK cells are incubated for an additional 12-20 h, as necessary, andmedium will be aspirated. The cells can be fixed with 3.7%paraformaldehyde in PBS. On the day of assay, fixed cells are washed andendogenous peroxidases inactivated with 0.35% H2 O2/PBS for 30 minutesat 37° C. The wells are washed again and incubated with 50 μl of 1.5mg/ml of mouse monoclonal anti-influenza A or anti-influenza BHaemagglutinin and incubated for 45 min at 37° C. The cells are washedfour times with washing buffer (PBS and 0.02% Tween-20), and incubatedwith 50 μl of 1:2000 dilution of goat anti-mouse IgG (H+L) HRPconjugated (BioRad, ref. 170-6516) for 45 min at 37° C. Plates arewashed as previously with washing buffer and 100 μl of BD OptEIATMBsubstrate (BD Biosciences, San Jose, Calif.) added to each well then theplate can be incubated at 37° C.

The enzymatic reaction can be stopped after 3-5 min by the addition of50 μL of 1 M of H2SO4 per well. Raw data is obtained by reading theabsorbance (OD) of each well at 450 nm for 0.1 sec with a Victor 3multilabel reader (PerkinElmer, Waltham, Mass.). Final ODs are obtainedby subtraction of the negative control (non-infected cells) OD from theinitial OD reading and the data analysed with GraphPadPrism4 (GraphPadSoftware Inc., La Jolla, Calif.) to calculate IC50 values (nonlinearregression (curve fit), Dose-response—inhibition, 4 parameter logistic).The IC₅₀ value is considered as the concentration of inhibitor thatreduced the absorbance at 450 nm by 50%, compared to a non-treatedinfected cell monolayer.

The in situ cell based ELISA can be performed as for hPIV-3 with minormodifications including differences between hPIV-1 and 3 tests such as:Primary antibody: Mouse monoclonal anti-hPIV-3 HN (Fitzgerald, cloneM02122321); Mouse monoclonal anti-hPIV-1 HN (LSbio, ref LS-C74109);hPIV-3 infection media: EMEM+2 mM glutamine; hPIV-1 infection media:EMEM+2 mM glutamine+TrypLE select 1.2%.

hPIV-3 NI and hPIV-1 NI IC50 Values for Compounds of the Invention

IC50 values for a number of compounds of the first aspect weredetermined by standard assay methods previously described in Guillon, Pet al, Nature Communications (2014). In the below tables are IC50 valuesfor prepared compounds wherein the chemistry ID aligns with thosereferences and compound structures provided in the experimentalcharacterisation section.

TABLE 5 hPIV-3 and hPIV-1 IC50 values for tested compounds Chemistry IDIC50 (μM) hPIV-3 NI (IC50 values) IE1172-78 2.47 IE1172-82 3.95IE1172-83 5.61 IE1172-87 77.35 IE1172-45 315 IE1172-102 >1000 IE1257-842.19 IE1398-33 1.97 IE832-8 54.43 IE832-12 51.85 IE832-17 148.5 IE832-206.28 IE832-26 67.67 IE832-27 106.1 IE832-31 22.92 IE832-37 2.4 IE889-343.23 IE889-52 5.85 IE927-60 13.13 IE927-67 114.3 IE984-5 1.38 IE1257-248.49 IE889-80 2.68 IE889-99 0.599 IE927-99 0.268 IE832-98 27.38 IE889-425.11 hPIV-1 NI (IC50 values) IE1172-78 75.15 IE1172-82 57.18 IE1172-8373.62 IE1172-87 92.79 IE1172-45 >1000 IE1172-102 793.4 IE1257-84 24.88IE1398-33 no data IE832-8 300.6 IE832-12 215.3 IE832-17 19.82 IE832-20192.7 IE832-26 95.49 IE832-27 301.9 IE832-31 303.5 IE832-37 200.6IE889-34 30.3 IE889-52 168.5 IE927-60 0.489 IE927-67 47.74 IE984-5 21.88IE1257-24 0.36 IE889-80 16.9 IE889-99 6.22 IE927-99 3.09 IE832-98 7.44IE889-42 0.159

The invention claimed is:
 1. A compound of formula (I), or apharmaceutically acceptable salt thereof:

wherein, R₁ is selected from the group consisting of COOH, or a saltthereof, C(O)NR₉R₁₀, C(O)OR₁₁ wherein R₉, R₁₀ and R₁₁ are independentlyselected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl; R₃ is selected from the group consisting ofoptionally substituted N-linked tetrazole, optionally substitutedN-linked indole, optionally substituted N-linked isoindole, optionallysubstituted N-linked benzotriazole, and N-linked triazole substituted atone or both ring carbon atoms having the below structure:

wherein, R₂₀ and R₂₁ are selected from the group consisting of hydrogen,hydroxyl, cyano, halo, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkylether,optionally substituted pyridyl and optionally substituted phenyl, withthe proviso that when R₂₀ is hydrogen and R₄ is AcHN then R₂₁ isselected from the group consisting of hydrogen, hydroxyl, cyano, halo,C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkylether, substituted pyridyl andsubstituted phenyl wherein substitution of pyridyl and phenyl isindependently with a moiety selected from the group consisting ofmethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,pentyl, isoamyl, 2-methylbutyl, 3-methylbutyl, hexyl, C₁-C₆ haloalkyl,C₁-C₆ alkylhydroxy, C₁-C₂ alkoxy, carboxy and salts thereof, C₄-C₆alkoxy, Cl, Br, I, and —CH₂OCH₃; and wherein at least one of R₂₀ and R₂₁is not hydrogen; R₄ is NHC(O)R₁₇ wherein R₁₇ is selected from the groupconsisting of C₁-C₆ alkyl, C₁-C₆ haloalkyl and C₃-C₆ cycloalkyl; R₆, R₇and R₈ are independently selected from the group consisting of OH, NH₂,C₁-C₆ alkyl, NR₁₈R₁₈′, C₁-C₆ alkoxy, —OC(O)R₁₈, —NH(C═O)R₁₈, andS(O)_(n)R₁₈, wherein n=0-2 and each R₁₈ and R₁₈′ are independentlyhydrogen or optionally substituted C₁-C₆ alkyl, and with the provisothat when R₄ is NHAc and R₃ is a triazole substituted only at the R₂₁position then the triazole is not substituted with propyl, substitutedpropyl, substituted tert-butyl or diethoxyalkyl.
 2. The compound ofclaim 1 wherein R₁ is COOH, or a salt thereof, or C(O)OR₁₁ wherein R₁₁is selected from methyl, ethyl and propyl.
 3. The compound of claim 1wherein R₃ is selected from the group consisting of:

wherein, R₂₀ and R₂₁ as defined in claim 1; R₂₂ is selected from thegroup consisting of hydrogen, C₁-C₆ alkyl and optionally substitutedphenyl; and R₂₃ and R₂₄ are independently selected from the groupconsisting of hydrogen, hydroxyl, cyano, halo, C₁-C₆ alkyl and C₁-C₆haloalkyl.
 4. The compound of claim 3 wherein when R₂₂ is optionallysubstituted phenyl then the substitution may be with a moiety selectedfrom the group consisting of C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆alkylhydroxy, C₁-C₆ alkoxy, halo, —C(O)OMe and —CH₂OCH₃.
 5. The compoundof claim 1 wherein R₃ is selected from the group consisting of:


6. The compound of claim 1 wherein R₄ is selected from the groupconsisting of:


7. The compound of claim 1 wherein R₄ is selected from the groupconsisting of —NHAc, —NHC(O)CH₂(CH₃)₂, —NHC(O)CF₃ and —NHC(O)CH₂CH₃. 8.The compound of claim 1 wherein R₆, R₇ and R₈ are independently selectedfrom OH and OAc.
 9. The compound of claim 1 wherein the compound offormula (I) is a compound of formula (II):

wherein, R₁, R₃, R₄, R₆, R₇ and R₈ are as described in any one of thepreceding claims.
 10. The compound of claim 1 wherein the compound offormula (I) is a compound selected from the group consisting of:

and analogues thereof wherein the C-2 carboxy group is in the protonatedform, sodium salt form or C₁-C₃ ester prodrug form and wherein the R₄position is substituted with any —NHC(O)R group wherein R is C₁-C₄ alkylor haloalkyl.
 11. A pharmaceutical composition comprising an effectiveamount of a compound of claim 1, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier, diluent and/orexcipient.
 12. A method of treating a disease, disorder or conditioncaused by parainfluenza virus in a patient including the step ofadministering an effective amount of a compound of claim 1 to thepatient.
 13. The method of claim 12 wherein the parainfluenza virus isselected from the group consisting of the hPIV-1, 2 and 3 virus.
 14. Themethod of claim 12 wherein the patient is a domestic or livestock animalor a human.
 15. A method of inhibiting the activity of a parainfluenzaviral haemagglutinin and/or neuraminidase enzyme by contacting theenzyme with a compound of claim 1, or a pharmaceutically effective saltthereof.